Tissue-specific expression and hybrid plant production

ABSTRACT

This disclosure concerns the use of endogenous plant RNAi machinery to preferentially or specifically reduce transgene expression. In some embodiments, the disclosure concerns specific reduction of transgene expression in male plant tissues, for example, to provide an economical male sterility system of hybrid seed production.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 14/577,887, filed Dec. 19, 2014, which claims the benefit ofthe filing date of U.S. Provisional Patent Application Ser. No.61/922,603, filed Dec. 31, 2013, the disclosures of which are herebyincorporated herein in their entirety by this reference.

TECHNICAL FIELD

The present disclosure relates to constructs and methods for the controlof gene expression in plants. Specific embodiments herein relate toconstructs and methods for utilizing the native RNA inhibition (RNAi)machinery of a plant cell to effect tissue-specific expression of a geneof interest, for example, in male plant tissues.

BACKGROUND

The development of hybrid crop varieties has enabled an increase in cropproductivity, mainly due to hybrid vigor and increased uniformity. Mostcrops show hybrid vigor, but commercial production of hybrids is onlyfeasible if a reliable and cost-effective pollination control system isavailable; hybrid seed production requires a system that preventsunwanted self-pollination.

Methods that can be used to prevent self-pollination include mechanicalremoval of anthers or male flowers, application of male-specificgametocides, and use of genetic cytoplasmic or nuclear-encoded malesterility. Mechanical removal is an expensive practice, which alsoundesirably reduces crop yield due to plant damage. With respect to theuse of cytoplasmic male-sterile (CMS) lines, these lines have a mutationin their mitochondrial genome, and thus the male sterility is inheritedas a dominant, maternally-transmitted trait. Cytoplasmic male sterilityrequires CMS mutants and nuclear restorer available in a given crop.Perez-Prat and van Lookeren Campagne (2002) Trends Plant Sci. 7:199-203.

In view of the foregoing considerations, the production of commercialhybrid corn seed typically utilizes the planting of male and femaleinbred lines in separate rows or blocks in an isolated field to reducethe possibility of contamination. The female inbred is then detasseledbefore pollen shed, which ensures cross-pollination by the male inbred.Hybrid seed is harvested and processed from the ears of thecross-pollinated female inbred. Manual or mechanical detasselingcontributes to the high cost of hybrid corn seed. Furthermore, hybridseeds generally have lower yields, which further results in lowerrevenues and profitability.

RNA interference (RNAi) is a process utilizing endogenous cellularpathways, whereby an interfering RNA (iRNA) molecule (e.g., a dsRNAmolecule) that is specific for a target gene sequence results in thedegradation of the mRNA encoded thereby. In recent years, RNAi has beenused to perform gene “knockdown” in a number of species and experimentalsystems; for example, C. elegans, plants, insect embryos, and cells intissue culture. See, e.g., Fire et al. (1998) Nature 391:806-11;Martinez et al. (2002) Cell 110:563-74; McManus and Sharp (2002) NatureRev. Genetics 3:737-47.

RNAi accomplishes degradation of mRNA through an endogenous pathwayincluding the DICER protein complex. DICER cleaves long dsRNA moleculesinto short double-stranded fragments of approximately 20 nucleotides.The inhibitory double-stranded RNA is unwound into two single-strandedRNAs: the passenger strand and the guide strand. The passenger strand isdegraded, and the guide strand is incorporated into the RNA-inducedsilencing complex (RISC). Post-transcriptional gene silencing(translational repression) occurs when the guide strand bindsspecifically to a complementary sequence of an mRNA molecule and inducescleavage by Argonaute, the catalytic component of the RISC complex.

Plant micro-RNAs (miRNAs) are typically produced from fold-backstructures having a partial double-stranded structure (e.g.,“hairpins”), and usually are nearly perfectly complementarity withtarget sites, which are found most commonly in protein-coding regions ofthe genome. As a result, plant miRNAs function generally to guide mRNAcleavage. Watson et al. (2005) FEBS Lett. 579:5982-7. In contrast,animal miRNAs contain relatively low levels of complementarity to theirtarget sites, and thus generally do not guide cleavage, but ratherfunction to repress expression at the translational or co-translationallevel. Watson et al. (2005), supra; Tomari and Zamore (2005) Genes Dev.19(5):517-29. Although miRNA sequences are not conserved between plantsand animals, the RNAi pathways that utilize these genes are highlysimilar. Millar and Waterhouse (2005) Funct. Integr. Genomics 5:129-35.For example, while the biogenesis of miRNAs in plants is accomplished bya different set of related enzymes than accomplish the biogenesis ofanimal miRNAs, the miRNA molecules themselves have a characteristicstructure that is capable of effecting mRNA cleavage or translationalrepression, depending on their degree of sequence complementarity to thetarget gene. Id.

In addition to miRNAs, plants also produce endogenous 21-25 nucleotidesmall inhibitory-RNAs (siRNAs). Most of these differ from miRNAs, inthat they arise from double-stranded RNA (rather than imperfectfold-back structures), which in some cases are generated by the activityof RNA-Dependent RNA Polymerases (RDRs).

Most plants contain four DICER-LIKE (DCL) proteins, one of which (DCL1)is necessary for maturation of most miRNA precursors. Kurihara andWatanabe (2004) Proc. Natl. Acad. Sci. USA 101:12753-8. Animal miRNAprecursor processing requires the sequential nucleolytic activity ofDROSHA and DICER. Lee et al. (2003) Nature 425:415-9. In animals,Exportin-5 (ExpS) regulates the transport of pre-miRNAs from the nucleusto the cytoplasm. Bohnsack et al. (2004) RNA 10:185-91.

Only RNA transcripts complementary to the siRNA and/or miRNA are cleavedand degraded by RNAi, and thus the knock-down of mRNA expression issequence-specific. The gene silencing effect of RNAi persists for daysand, under experimental conditions, can lead to a decline in abundanceof the targeted transcript of 90% or more, with consequent reduction inlevels of the corresponding protein.

BRIEF SUMMARY

Described herein are the compositions and methods of a novel plant smallRNA (sRNA)-mediated approach for transgenic plant (e.g., hybrids)production. The approach utilizes sRNAs (e.g., endogenous siRNAs andmiRNAs) that are expressed in specific tissues for targeted repressionor knockdown of (trans) gene expression. In some embodiments, a targetsite of an sRNA is fused within a transgene sequence that confersherbicide tolerance. Tissue-specific expression of sRNA (e.g.,endogenous sRNAs) in the tissue results in down-regulation of thetransgene, which confers sensitivity to the herbicide upon the specifictissue, while other plant tissues remain tolerant.

In some embodiments, sRNAs are specifically expressed in male tissue ofa plant, so as to repress/knockdown the expression of a transgene andthereby eliminate the need for manual detasseling. Particularembodiments include the sRNA-mediated tissue-specific expression of anytransgene in plants that allows for timed induction of male sterility.In examples, the transgene is an herbicide tolerance gene, andapplication of the herbicide to the plant before or during tassel stageprevents tassel development and self-pollination. In some embodiments,sRNAs are specifically or preferentially expressed in, for example, theroot tissue of a plant, so as to specifically or preferentially reduceexpression of a transgene in the tissue.

Described herein are nucleic acid expression constructs, whichconstructs comprise a gene of interest having an internal target sitefor at least one sRNA molecule. In embodiments, RNA may be transcribedfrom the gene of interest in vivo when the construct is introduced intoa plant cell, which RNA is then degraded by an RNAi mechanism under thecontrol of the sRNA molecule(s). In particular embodiments, the gene ofinterest is an agronomic trait gene, an herbicide resistance gene or aselectable marker gene. In particular embodiments, the internal sRNAtargeting site is, for example, 20-25 nucleotides in length.

Also described herein are transgenic plant cells, plant tissues, andplants comprising at least one of the foregoing nucleic acid expressionconstructs. In particular embodiments, the sRNA molecule isdifferentially expressed in different plant cells and tissues. Forexample, the gene (e.g., transgene) encoding the sRNA molecule may beoperably linked to a non-constitutive promoter (e.g., a tissue-specificpromoter). In particular embodiments, the sRNA is an endogenous sRNA ofthe plant cell. In certain examples, the sRNA is an endogenous sRNA thatis expressed predominantly in male plant tissues, which sRNA directsdegradation specifically in male tissues of RNA transcribed from thegene of interest, wherein the expression of RNA transcribed from thegene of interest in other tissues is essentially unaffected.

Further described herein are pollination control systems comprising theforegoing constructs and/or plant cells, plant tissues, and plantscomprising such constructs.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 includes a diagram of particular embodiments, wherein maletissue-specific herbicide susceptibility is conferred upon a transgeniccorn plant as part of a pollination control system.

FIG. 2 includes data showing the transient expression of AAD1 inimmature corn embryos.

FIGS. 3A and 3B include data showing the expression of AAD1 in tassel.

FIGS. 4A and 4B include data showing the expression of AAD1 in leaf.

FIGS. 5A and 5B include data showing the expression of AAD1 RNA in leaf(FIG. 5A) and tassel (FIG. 5B).

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, asdefined in 37 C.F.R. § 1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood to beincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NO:1 shows, in the 5′ to 3′ direction, an exemplary miR156 RNA:

UGACAGAAGAGAGUGAGCAC

SEQ ID NO:2 shows, in the 5′ to 3′ direction, an exemplary miR529 RNA:

AGAAGAGAGAGAGUACAGCCU

SEQ ID NO:3 shows an exemplary miR319 RNA:

UUGGACUGAAGGGUGCUCCC

SEQ ID NO:4 shows a tsh4 miR529-156 DNA target site:

GACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCA

SEQ ID NO:5 shows an exemplary expression cassette comprising an AAD-1v3 coding region (underlined), followed by a fragment comprising a 3′UTR from a maize peroxidase 5 gene, ZmPer5 3′UTR (italics):

ATGGCTCATGCTGCCCTCAGCCCTCTCTCCCAACGCTTTGAGAGAATAGCTGTCCAGCCACTCACTGGTGTCCTTGGTGCTGAGATCACTGGAGTGGACTTGAGGGAACCACTTGATGACAGCACCTGGAATGAGATATTGGATGCCTTCCACACTTACCAAGTCATCTACTTTCCTGGCCAAGCAATCACCAATGAGCAGCACATTGCATTCTCAAGAAGGTTTGGACCAGTTGATCCAGTGCCTCTTCTCAAGAGCATTGAAGGCTATCCAGAGGTTCAGATGATCCGCAGAGAAGCCAATGAGTCTGGAAGGGTGATTGGTGATGACTGGCACACAGACTCCACTTTCCTTGATGCACCTCCAGCTGCTGTTGTGATGAGGGCCATAGATGTTCCTGAGCATGGCGGAGACACTGGGTTCCTTTCAATGTACACAGCTTGGGAGACCTTGTCTCCAACCATGCAAGCCACCATCGAAGGGCTCAACGTTGTGCACTCTGCCACACGTGTGTTCGGTTCCCTCTACCAAGCACAGAACCGTCGCTTCAGCAACACCTCAGTCAAGGTGATGGATGTTGATGCTGGTGACAGAGAGACAGTCCATCCCTTGGTTGTGACTCATCCTGGCTCTGGAAGGAAAGGCCTTTATGTGAATCAAGTCTACTGTCAGAGAATTGAGGGCATGACAGATGCAGAATCAAAGCCATTGCTTCAGTTCCTCTATGAGCATGCCACCAGATTTGACTTCACTTGCCGTGTGAGGTGGAAGAAAGACCAAGTCCTTGTCTGGGACAACTTGTGCACCATGCACCGTGCTGTTCCTGACTATGCTGGCAAGTTCAGATACTTGACTCGCACCACAGTTGGTGGAGTTAGGCCTGCCCGCTGAGTAGTTAGCTTAATCACCTAGAGCTCGGTAACCTTTAAACTGAGGGCACTGAAGTCGCTTGATGTGCTGAATTGTTTGTGATGTTGGTGGCGTATTTTGTTTAAATAAGTAAGCATGGCTGTGATTTTATCATATGATCGATCTTTGGGGTTTTATTTAACACATTGTAAAATGTGTATCTATTAATAACTCAATGTATAAGATGTGTTCATTCTTCGGTTGCCATAGATCTGCTTATTTGACCTGTGATGTTTTGACTCCAAAAACCAAAATCACAACTCAATAAACTCATGGAATATGTCCACCTGTTTCTTGAAGAGTTCATCTACCATTCCAGTTGGCATTTATCAGTGTTGCAGCGGCGCTGTGCTTTGTAACATAACAATTGTTACGGCATATATCCAA

SEQ ID NO:6 shows an exemplary polynucleotide comprising the AAD-1coding region (underlined), followed by a native tsh4 miR529-156 targetsite (double underlined) positioned between the end of the AAD-1 codingregion and the ZmPer5 3′UTR (italics):

ATGGCTCATGCTGCCCTCAGCCCTCTCTCCCAACGCTTTGAGAGAATAGCTGTCCAGCCACTCACTGGTGTCCTTGGTGCTGAGATCACTGGAGTGGACTTGAGGGAACCACTTGATGACAGCACCTGGAATGAGATATTGGATGCCTTCCACACTTACCAAGTCATCTACTTTCCTGGCCAAGCAATCACCAATGAGCAGCACATTGCATTCTCAAGAAGGTTTGGACCAGTTGATCCAGTGCCTCTTCTCAAGAGCATTGAAGGCTATCCAGAGGTTCAGATGATCCGCAGAGAAGCCAATGAGTCTGGAAGGGTGATTGGTGATGACTGGCACACAGACTCCACTTTCCTTGATGCACCTCCAGCTGCTGTTGTGATGAGGGCCATAGATGTTCCTGAGCATGGCGGAGACACTGGGTTCCTTTCAATGTACACAGCTTGGGAGACCTTGTCTCCAACCATGCAAGCCACCATCGAAGGGCTCAACGTTGTGCACTCTGCCACACGTGTGTTCGGTTCCCTCTACCAAGCACAGAACCGTCGCTTCAGCAACACCTCAGTCAAGGTGATGGATGTTGATGCTGGTGACAGAGAGACAGTCCATCCCTTGGTTGTGACTCATCCTGGCTCTGGAAGGAAAGGCCTTTATGTGAATCAAGTCTACTGTCAGAGAATTGAGGGCATGACAGATGCAGAATCAAAGCCATTGCTTCAGTTCCTCTATGAGCATGCCACCAGATTTGACTTCACTTGCCGTGTGAGGTGGAAGAAAGACCAAGTCCTTGTCTGGGACAACTTGTGCACCATGCACCGTGCTGTTCCTGACTATGCTGGCAAGTTCAGATACTTGACTCGCACCACAGTTGGTGGAGTTAGGCCTGCCCGCTGAGTAGTTAGCTTAATCACCTAGAGCTCgACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCaGGTAACCTTTAAACTGAGGGCACTGAAGTCGCTTGATGTGCTGAATTGTTTGTGATGTTGGTGGCGTATTTTGTTTAAATAAGTAAGCATGGCTGTGATTTTATCATATGATCGATCTTTGGGGTTTTATTTAACACATTGTAAAATGTGTATCTATTAATAACTCAATGTATAAGATGTGTTCATTCTTCGGTTGCCATAGATCTGCTTATTTGACCTGTGATGTTTTGACTCCAAAAACCAAAATCACAACTCAATAAACTCATGGAATATGTCCACCTGTTTCTTGAAGAGTTCATCTACCATTCCAGTTGGCATTTATCAGTGTTGCAGCGGCGCTGTGCTTTGTAACATAACAATTGTTACGGCATATATCCAA

SEQ ID NO:7 shows an exemplary modified tsh4 target site comprising anative miR529 target site with a mutated miR156 target site:

GACTCAGGCTGTACTCTCTTACTTCACAAAGTACTCA

SEQ ID NO:8 shows a further exemplary modified tsh4 target sitecomprising a native miR529 target site with a mutated miR156 targetsite:

GACTCAGGCTGTACTCTCTCTCTTCACAAAGTACTCA

SEQ ID NO:9 shows an exemplary polynucleotide comprising the AAD-1coding region (underlined), followed by an exemplary modified tsh4target site (double underlined) comprising a native miR529 target sitewith a mutated miR156 target site, positioned between the end of theAAD-1 coding region and the ZmPer5 3′UTR (italics):

ATGGCTCATGCTGCCCTCAGCCCTCTCTCCCAACGCTTTGAGAGAATAGCTGTCCAGCCACTCACTGGTGTCCTTGGTGCTGAGATCACTGGAGTGGACTTGAGGGAACCACTTGATGACAGCACCTGGAATGAGATATTGGATGCCTTCCACACTTACCAAGTCATCTACTTTCCTGGCCAAGCAATCACCAATGAGCAGCACATTGCATTCTCAAGAAGGTTTGGACCAGTTGATCCAGTGCCTCTTCTCAAGAGCATTGAAGGCTATCCAGAGGTTCAGATGATCCGCAGAGAAGCCAATGAGTCTGGAAGGGTGATTGGTGATGACTGGCACACAGACTCCACTTTCCTTGATGCACCTCCAGCTGCTGTTGTGATGAGGGCCATAGATGTTCCTGAGCATGGCGGAGACACTGGGTTCCTTTCAATGTACACAGCTTGGGAGACCTTGTCTCCAACCATGCAAGCCACCATCGAAGGGCTCAACGTTGTGCACTCTGCCACACGTGTGTTCGGTTCCCTCTACCAAGCACAGAACCGTCGCTTCAGCAACACCTCAGTCAAGGTGATGGATGTTGATGCTGGTGACAGAGAGACAGTCCATCCCTTGGTTGTGACTCATCCTGGCTCTGGAAGGAAAGGCCTTTATGTGAATCAAGTCTACTGTCAGAGAATTGAGGGCATGACAGATGCAGAATCAAAGCCATTGCTTCAGTTCCTCTATGAGCATGCCACCAGATTTGACTTCACTTGCCGTGTGAGGTGGAAGAAAGACCAAGTCCTTGTCTGGGACAACTTGTGCACCATGCACCGTGCTGTTCCTGACTATGCTGGCAAGTTCAGATACTTGACTCGCACCACAGTTGGTGGAGTTAGGCCTGCCCGCTGAGTAGTTAGCTTAATCACCTAGAGCTCgACTCAGGCTGTACTCTCTTACTTCACAAAGTACTCaGGTAACCTTTAAACTGAGGGCACTGAAGTCGCTTGATGTGCTGAATTGTTTGTGATGTTGGTGGCGTATTTTGTTTAAATAAGTAAGCATGGCTGTGATTTTATCATATGATCGATCTTTGGGGTTTTATTTAACACATTGTAAAATGTGTATCTATTAATAACTCAATGTATAAGATGTGTTCATTCTTCGGTTGCCATAGATCTGCTTATTTGACCTGTGATGTTTTGACTCCAAAAACCAAAATCACAACTCAATAAACTCATGGAATATGTCCACCTGTTTCTTGAAGAGTTCATCTACCATTCCAGTTGGCATTTATCAGTGTTGCAGCGGCGCTGTGCTTTGTAACATAACAATTGTTACGGCATATATCCAA

SEQ ID NO:10 shows an exemplary polynucleotide comprising the AAD-1coding region (underlined), followed by a further exemplary modifiedtsh4 target site (double underlined) comprising a native miR529 targetsite with a mutated miR156 target site, positioned between the end ofthe AAD-1 coding region and the ZmPer5 3′UTR (italics):

ATGGCTCATGCTGCCCTCAGCCCTCTCTCCCAACGCTTTGAGAGAATAGCTGTCCAGCCACTCACTGGTGTCCTTGGTGCTGAGATCACTGGAGTGGACTTGAGGGAACCACTTGATGACAGCACCTGGAATGAGATATTGGATGCCTTCCACACTTACCAAGTCATCTACTTTCCTGGCCAAGCAATCACCAATGAGCAGCACATTGCATTCTCAAGAAGGTTTGGACCAGTTGATCCAGTGCCTCTTCTCAAGAGCATTGAAGGCTATCCAGAGGTTCAGATGATCCGCAGAGAAGCCAATGAGTCTGGAAGGGTGATTGGTGATGACTGGCACACAGACTCCACTTTCCTTGATGCACCTCCAGCTGCTGTTGTGATGAGGGCCATAGATGTTCCTGAGCATGGCGGAGACACTGGGTTCCTTTCAATGTACACAGCTTGGGAGACCTTGTCTCCAACCATGCAAGCCACCATCGAAGGGCTCAACGTTGTGCACTCTGCCACACGTGTGTTCGGTTCCCTCTACCAAGCACAGAACCGTCGCTTCAGCAACACCTCAGTCAAGGTGATGGATGTTGATGCTGGTGACAGAGAGACAGTCCATCCCTTGGTTGTGACTCATCCTGGCTCTGGAAGGAAAGGCCTTTATGTGAATCAAGTCTACTGTCAGAGAATTGAGGGCATGACAGATGCAGAATCAAAGCCATTGCTTCAGTTCCTCTATGAGCATGCCACCAGATTTGACTTCACTTGCCGTGTGAGGTGGAAGAAAGACCAAGTCCTTGTCTGGGACAACTTGTGCACCATGCACCGTGCTGTTCCTGACTATGCTGGCAAGTTCAGATACTTGACTCGCACCACAGTTGGTGGAGTTAGGCCTGCCCGCTGAGTAGTTAGCTTAATCACCTAGAGCTCgACTCAGGCTGTACTCTCTCTCTTCACAAAGTACTCaGGTAACCTTTAAACTGAGGGCACTGAAGTCGCTTGATGTGCTGAATTGTTTGTGATGTTGGTGGCGTATTTTGTTTAAATAAGTAAGCATGGCTGTGATTTTATCATATGATCGATCTTTGGGGTTTTATTTAACACATTGTAAAATGTGTATCTATTAATAACTCAATGTATAAGATGTGTTCATTCTTCGGTTGCCATAGATCTGCTTATTTGACCTGTGATGTTTTGACTCCAAAAACCAAAATCACAACTCAATAAACTCATGGAATATGTCCACCTGTTTCTTGAAGAGTTCATCTACCATTCCAGTTGGCATTTATCAGTGTTGCAGCGGCGCTGTGCTTTGTAACATAACAATTGTTACGGCATATATCCAA

SEQ ID NO:11 shows an artificial miRNA, referred to herein asCMSRF9973.1:

GGATCCCAGCAGCAGCCACAGCAAAATTTGGTTTGGGATAGGTAGGTGTTATGTTAGGTCTGGTTTTTTGGCTGTAGCAGCAGCAGAGAAGAGAGAGAGTACAGCCTCAGGAGATTCAGTTTGAAGCTGGACTTCACTTTTGCCTCTCTAGGCTCTACACTCTCTCTTCTTTCCTGCTGCTAGGCTGTTCTGTGGAAGTTTGCAGAGTTTATATTATGGGTTTAATCGTCCATGGCATCAGCATCAGCAG CATTTATGAGCTCGGTAACC

SEQ ID NO:12 shows an SCBV promoter, utilized in certain examplesherein:

TCGGAAGTTGAAGACAAAGAAGGTCTTAAATCCTGGCTAGCAACACTGAACTATGCCAGAAACCACATCAAAGCATATCGGCAAGCTTCTTGGCCCATTATATCCAAAGACCTCAGAGAAAGGTGAGCGAAGGCTCAATTCAGAAGATTGGAAGCTGATCAATAGGATCAAGACAATGGTGAGAACGCTTCCAAATCTCACTATTCCACCAGAAGATGCATACATTATCATTGAAACAGATGCATGTGCAACTGGATGGGGAGCAGTATGCAAGTGGAAGAAAAACAAGGCAGACCCAAGAAATACAGAGCAAATCTGTAGGTATGCCAGTGGAAAATTTGATAAGCCAAAAGGAACCTGTGATGCAGAAATCTATGGGGTTATGAATGGCTTAGAAAAGATGAGATTGTTCTACTTGGACAAAAGAGAGATCACAGTCAGAACTGACAGTAGTGCAATCGAAAGGTTCTACAACAAGAGTGCTGAACACAAGCCTTCTGAGATCAGATGGATCAGGTTCATGGACTACATCACTGGTGCAGGACCAGAGATAGTCATTGAACACATAAAAGGGAAGAGCAATGGTTTAGCTGACATCTTGTCCAGGCTCAAAGCCAAATTAGCTCAGAATGAACCAACGGAAGAGATGATCCTGCTTACACAAGCCATAAGGGAAGTAATTCCTTATCCAGATCATCCATACACTGAGCAACTCAGAGAATGGGGAAACAAAATTCTGGATCCATTCCCCACATTCAAGAAGGACATGTTCGAAAGAACAGAGCAAGCTTTTATGCTAACAGAGGAACCAGTTCTACTCTGTGCATGCAGGAAGCCTGCAATTCAGTTAGTGTCCAGAACATCTGCCAACCCAGGAAGGAAATTCTTCAAGTGCGCAATGAACAAATGCCATTGCTGGTACTGGGCAGATCTCATTGAAGAACACATTCAAGACAGAATTGATGAATTTCTCAAGAATCTTGAAGTTCTGAAGACCGGTGGCGTGCAAACAATGGAGGAGGAACTTATGAAGGAAGTCACCAAGCTGAAGATAGAAGAGCAGGAGTTCGAGGAATACCAGGCCACACCAAGGGCTATGTCGCCAGTAGCCGCAGAAGATGTGCTAGATCTCCAAGACGTAAGCAATGACGATTGAGGAGGCATTGACGTCAGGGATGACCGCAGCGGAGAGTACTGGGCCCATTCAGTGGATGCTCCACTGAGTTGTATTATTGTGTGCTTTTCGGACAAGTGTGCTGTCCACTTTCTTTTGGCACCTGTGCCACTTTATTCCTTGTCTGCCACGATGCCTTTGCTTAGCTTGTAAGCAAGGATCGCAGTGCGTGTGTGACACCACCCCCCTTCCGACGCTCTGCCTATATAAGGCACCGTCTGTAAGCTCTTACGATCATCGGTAGTTCACCAAGGC

SEQ ID NO:13 shows a synthetic 5′ UTR, utilized in certain examplesherein:

CTGAAGGCTCGACAAGGCAGTCCACGGAGGAGCTGATATTTGGTGGACAAGCTGTGGATAGGAGCAACCCTATCCCTAATATACCAGCACCACCAAGTCAGGGCAATCCCCAGATCACCCCAGCAGATTCGAAGAAGGTACAGTACACACACATGTATATATGTATGATGTATCCCTTCGATCGAAGGCATGCCTTGGTATAATCACTGAGTAGTCATTTTATTACTTTGTTTTGACAAGTCAGTAGTTCATCCATTTGTCCCATTTTTTCAGCTTGGAAGTTTGGTTGCACTGGCCTTGGTCTAATAACTGAGTAGTCATTTTATTACGTTGTTTCGACAAGTCAGTAGCTCATCCATCTGTCCCATTTTTTCAGCTAGGAAGTTTGGTTGCACTGGCCTTGGACTAATAACTGATTAGTCATTTTATTACATTGTTTCGACAAGTCAGTAGCTCATCCATCTGTCCCATTTTTCAGCTAGGAAGTTCGGATCTGGGGCCATTTGTTCCAGGCACGGGATAAGCATTCAG

SEQ ID NO:14 shows a maize Invertase (INV) gene (GenBank™ Accession No.U16123).

SEQ ID NO:15 shows a maize Elongation Factor 1α (EF1α) gene (GenBank™Accession No. AF136823.1).

SEQ ID NOs:16-31 show oligonucleotides utilized in certain examplesherein.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Embodiments herein exploit tissue-specific expression of sRNAs torepress/knockdown the expression of a transgene in the specific tissue.sRNAs have been shown to regulate diverse developmental processes,including organ separation, polarity, and identity, and to modulatetheir own biogenesis and function. In embodiments herein, the precisionand specificity of sRNA-mediated gene regulation is utilized toaccomplish the tissue-specific expression of a transgene. In examples, atarget site for a tissue-specific sRNA (e.g., an endogenoustissue-specific sRNA) is engineered into the transgene to reduce oreliminate expression of the transgene in the tissue. Embodiments hereinmay be utilized to target the expression of any transgene to anyparticular tissue(s).

Particular embodiments herein are useful, for example, in preferentiallyrepressing expression of an herbicide selection gene in male tissues(e.g., tassel, pollen, anther, and stamen) to induce male sterility viaherbicide application. Examples of this novel cellular engineeringapproach will save enormous resources, for example, by reducing oreliminating the need to employ expensive manual detasseling proceduresduring hybrid seed production. In addition, some examples avoid yieldloss that is due to plant damage occurring during detasseling processes.

II. Abbreviations

-   -   dsRNA double-stranded ribonucleic acid    -   GI growth inhibition    -   NCBI National Center for Biological Information    -   gDNA genomic DNA    -   iRNA inhibitory ribonucleic acid    -   ORF open reading frame    -   RNAi ribonucleic acid interference    -   miRNA micro inhibitory ribonucleic acid    -   sRNA small ribonucleic acid    -   siRNA small inhibitory ribonucleic acid, or short, interfering        ribonucleic acid    -   hpRNA hairpin ribonucleic acid    -   UTR untranslated region    -   PCR polymerase chain reaction    -   RISC RNA-induced Silencing Complex

III. Terms

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Backcrossing: Backcrossing methods may be used to introduce a nucleicacid sequence into plants. The backcrossing technique has been widelyused for decades to introduce new traits into plants. N. Jensen, Ed.Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typicalbackcross protocol, the original variety of interest (recurrent parent)is crossed to a second variety (non-recurrent parent) that carries agene of interest to be transferred. The resulting progeny from thiscross are then crossed again to the recurrent parent, and the process isrepeated until a plant is obtained wherein essentially all of thedesired morphological and physiological characteristics of the recurrentplant are recovered in the converted plant, in addition to thetransferred gene from the non-recurrent parent.

Expression: As used herein, “expression” of a coding sequence (forexample, a gene or a transgene) refers to the process by which the codedinformation of a nucleic acid transcriptional unit (including, e.g.,genomic DNA or cDNA) is converted into an operational, non-operational,or structural part of a cell, often including the synthesis of aprotein. Gene expression can be influenced by external signals; forexample, exposure of a cell, tissue, or organism to an agent thatincreases or decreases gene expression. Expression of a gene can also beregulated anywhere in the pathway from DNA to RNA to protein. Regulationof gene expression occurs, for example, through controls acting ontranscription, translation, RNA transport and processing, degradation ofintermediary molecules such as mRNA, or through activation,inactivation, compartmentalization, or degradation of specific proteinmolecules after they have been made, or by combinations thereof. Geneexpression can be measured at the RNA level or the protein level by anymethod known in the art, including, without limitation, Northern blot,RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activityassay(s).

Genetic material: As used herein, the term “genetic material” includesall genes, and nucleic acid molecules, such as DNA and RNA.

Inhibition: As used herein, the term “inhibition,” when used to describean effect on a coding sequence (for example, a gene), refers to ameasurable decrease in the cellular level of mRNA transcribed from thecoding sequence and/or peptide, polypeptide, or protein product of thecoding sequence. In some examples, expression of a coding sequence maybe inhibited such that expression is essentially eliminated (e.g.,expression is reduced to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% of its expression in a control cell).

Isolated: An “isolated” biological component (such as a nucleic acid orprotein) has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs (i.e., otherchromosomal and extra-chromosomal DNA and RNA, and proteins), whileeffecting a chemical or functional change in the component (e.g., anucleic acid may be isolated from a chromosome by breaking chemicalbonds connecting the nucleic acid to the remaining DNA in thechromosome). Nucleic acid molecules and proteins that have been“isolated” include nucleic acid molecules and proteins purified bystandard purification methods, wherein there has been a chemical orfunctional change in the nucleic acid or protein. The term also embracesnucleic acids and proteins prepared by recombinant expression in a hostcell, as well as chemically-synthesized nucleic acid molecules,proteins, and peptides.

Nucleic acid molecule: As used herein, the term “nucleic acid molecule”may refer to a polymeric form of nucleotides, which may include bothsense and anti-sense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. A nucleotide may refer to aribonucleotide, deoxyribonucleotide, or a modified form of either typeof nucleotide. A “nucleic acid molecule” as used herein is synonymouswith “nucleic acid” and “polynucleotide.” A nucleic acid molecule isusually at least 10 bases in length, unless otherwise specified. Thenucleotide sequence of a nucleic acid molecule is read from the 5′ tothe 3′ end of the molecule by convention. The “complement” of anucleotide sequence refers to the sequence, from 5′ to 3′, of thenucleobases which form base pairs with the nucleobases of the nucleotidesequence (i.e., A-T/U, and G-C). The “reverse complement” of a nucleicacid sequence refers to the sequence, from 3′ to 5′, of the nucleobaseswhich form base pairs with the nucleobases of the nucleotide sequence.

“Nucleic acid molecules” include single- and double-stranded forms ofDNA; single-stranded forms of RNA; and double-stranded forms of RNA(dsRNA). The term “nucleotide sequence” or “nucleic acid sequence”refers to both the sense and antisense strands of a nucleic acid aseither individual single strands or in the duplex. The term “ribonucleicacid” (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA(double-stranded RNA), sRNA (small RNA), siRNA (small interfering RNA),miRNA (micro-RNA), hpRNA (hairpin RNA), mRNA (messenger RNA), tRNA(transfer RNA), and cRNA (complementary RNA). The term “deoxyribonucleicacid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. Theterm “polynucleotide” will be understood by those in the art as astructural term that is defined by its nucleotide sequence, and thatincludes genomic sequences, ribosomal RNA sequences, transfer RNAsequences, messenger RNA sequences, operon sequences, and smallerengineered nucleotide sequences.

Small RNA (sRNA): sRNAs are non-coding RNAs that regulate geneexpression by pairing to the message of protein-coding genes to guidemRNA cleavage or repression of productive translation. Dugas and Bartel(2004) Curr. Opin. Plant Biolo. 7:512-20. As used herein, the term“small RNA” (sRNA) includes, for example, microRNA (miRNA), smallinterfering RNA (siRNA), trans-acting small interfering RNA (tasiRNA),and other sRNAs guiding cleavage, translational repression, and/or genesilencing.

Small interfering RNA (siRNA): RNA of approximately 21-25 nucleotidesthat is processed from a dsRNA by a DICER enzyme (in animals) or a DCLenzyme (in plants). The initial DICER or DCL products aredouble-stranded, in which the two strands are typically 21-25nucleotides in length and contain two unpaired bases at each 3′ end. Theindividual strands within the double stranded siRNA structure areseparated, and typically one of the siRNAs then are associated with amulti-subunit complex, the RNAi-induced silencing complex (RISC). Atypical function of the siRNA is to guide RISC to the target based onbase-pair complementarity.

Target nucleic acid (to be inhibited): As used herein, the term“target,” in reference to a polynucleotide, refers to any nucleic acidcontaining a sequence that interacts with a miRNA or siRNA, or that hasthe potential to yield a sequence that interacts with a miRNA or siRNA(for example, through transcription of a locus). The target can be acellular nucleic acid, such as an mRNA that encodes an essential ornonessential protein, or a foreign nucleic acid, such as a virus-derivedor transgene-derived RNA molecule. The target can be a DNA sequencecorresponding to a promoter, or a sequence corresponding to anyexpressed region of a genome, for instance.

Oligonucleotide: An oligonucleotide is a short nucleic acid molecule.Oligonucleotides may be formed by cleavage of longer nucleic acidsegments, or by polymerizing individual nucleotide precursors. Automatedsynthesizers allow the synthesis of oligonucleotides up to severalhundred base pairs in length. Because oligonucleotides may bind to acomplementary nucleotide sequence, they may be used as probes fordetecting DNA or RNA. Oligonucleotides composed of DNA(oligodeoxyribonucleotides) may be used in PCR, a technique for theamplification of small DNA sequences. In PCR, the oligonucleotide istypically referred to as a “primer,” which allows a DNA polymerase toextend the oligonucleotide and replicate the complementary strand.

A nucleic acid molecule may include either or both naturally occurringand modified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages. Nucleic acid molecules maybe modified chemically or biochemically, or may contain non-natural orderivatized nucleotide bases, as will be readily appreciated by those ofskill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, inter-nucleotide modifications (e.g.,uncharged linkages: for example, methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.; charged linkages: for example,phosphorothioates, phosphorodithioates, etc.; pendent moieties: forexample, peptides; intercalators: for example, acridine, psoralen, etc.;chelators; alkylators; and modified linkages: for example, alphaanomeric nucleic acids, etc.). The term “nucleic acid molecule” alsoincludes any topological conformation, including single-stranded,double-stranded, partially duplexed, triplexed, hairpinned, circular,and padlocked conformations.

As used herein with respect to DNA, the term “coding sequence” refers toa nucleotide sequence that is transcribed into RNA when placed under thecontrol of appropriate regulatory sequences. A “protein coding sequence”is a nucleotide sequence (DNA or RNA) that is ultimately translated intoa polypeptide, via transcription and mRNA. With respect to RNA, the term“coding sequence” refers to a nucleotide sequence that is translatedinto a peptide, polypeptide, or protein. The boundaries of a codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. Coding sequencesinclude, but are not limited to: genomic DNA; cDNA; EST; and recombinantnucleotide sequences.

Genome: As used herein, the term “genome” refers to chromosomal DNAfound within the nucleus of a cell, and also refers organelle DNA foundwithin subcellular components of the cell. In some embodiments of theinvention, a DNA molecule may be introduced into a plant cell such thatthe DNA molecule is integrated into the genome of the plant cell. Inthese and further embodiments, the DNA molecule may be either integratedinto the nuclear DNA of the plant cell, or integrated into the DNA ofthe chloroplast or mitochondrion of the plant cell.

Endogenous: The term “endogenous,” as applied to nucleic acids (e.g.,polynucleotides, DNA, RNA, and genes) herein, refers to one or morenucleic acid(s) that are normally (e.g., in a wild-type cell of the sametype and species) present within their specific environment or context.For example, an endogenous gene is one that is normally found in theparticular cell in question and in the same context (e.g., with regardto regulatory sequences). Endogenous nucleic acids can be distinguishedfrom exogenous and/or heterologous, for example and without limitation,by detection in the latter of sequences that are consequent withrecombination from bacterial plasmid; identification of atypical codonpreferences; and amplification of atypical sequences in a PCR reactionfrom primers characterized in a wild-type cell.

Exogenous: The term “exogenous,” as applied to nucleic acids herein,refers to one or more nucleic acid(s) that are not normally presentwithin their specific environment or context. For example, if a hostcell is transformed with a nucleic acid that does not occur in theuntransformed host cell in nature, then that nucleic acid is exogenousto the host cell. The term exogenous, as used herein, also refers to oneor more nucleic acid(s) that are identical in sequence to a nucleic acidalready present in a host cell, but that are located in a differentcellular or genomic context than the nucleic acid with the same sequencealready present in the host cell. For example, a nucleic acid that isintegrated in the genome of the host cell in a different location than anucleic acid with the same sequence is normally integrated in the genomeof the host cell is exogenous to the host cell. Furthermore, a nucleicacid (e.g., a DNA molecule) that is present in a plasmid or vector inthe host cell is exogenous to the host cell when a nucleic acid with thesame sequence is only normally present in the genome of the host cell.

Heterologous: The term “heterologous,” as applied to nucleic acids(e.g., polynucleotides, DNA, RNA, and genes) herein, means of differentorigin. For example, if a host cell is transformed with a nucleic acidthat does not occur in the untransformed host cell in nature, then thatnucleic acid is heterologous (and exogenous) to the host cell.Furthermore, different elements (e.g., promoter, enhancer, codingsequence, terminator, etc.) of a transforming nucleic acid may beheterologous to one another and/or to the transformed host. The termheterologous, as used herein, may also be applied to one or more nucleicacid(s) that are identical in sequence to a nucleic acid already presentin a host cell, but that are now linked to different additionalsequences and/or are present at a different copy number, etc.

Sequence identity: The term “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences, and amino acid sequences) over a comparisonwindow, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, e.g.,Altschul et al. (1990) J Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, Md.), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn)program may be employed using the default parameters. Nucleic acidsequences with even greater similarity to the reference sequences willshow increasing percentage identity when assessed by this method.

Specifically hybridizable/specifically complementary: As used herein,the terms “specifically hybridizable” and “specifically complementary”are terms that indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the nucleic acid molecule anda target nucleic acid molecule. Hybridization between two nucleic acidmolecules involves the formation of an anti-parallel alignment betweenthe nucleic acid sequences of the two nucleic acid molecules. The twomolecules are then able to form hydrogen bonds with corresponding baseson the opposite strand to form a duplex molecule that, if it issufficiently stable, is detectable using methods well known in the art.A nucleic acid molecule need not be 100% complementary to its targetsequence to be specifically hybridizable. However, the amount ofsequence complementarity that must exist for hybridization to bespecific is a function of the hybridization conditions used.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, N Y, 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, NY, 1995.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 20% mismatch betweenthe hybridization molecule and a homologous sequence within the targetnucleic acid molecule. “Stringent conditions” include further particularlevels of stringency. Thus, as used herein, “moderate stringency”conditions are those under which molecules with more than 20% sequencemismatch will not hybridize; conditions of “high stringency” are thoseunder which sequences with more than 10% mismatch will not hybridize;and conditions of “very high stringency” are those under which sequenceswith more than 5% mismatch will not hybridize.

The following are representative, non-limiting hybridization conditions.

High Stringency condition (detects sequences that share at least 90%sequence identity): Hybridization in 5×SSC buffer at 65° C. for 16hours; wash twice in 2×SSC buffer at room temperature for 15 minuteseach; and wash twice in 0.5×SSC buffer at 65° C. for 20 minutes each.

Moderate Stringency condition (detects sequences that share at least 80%sequence identity): Hybridization in 5×-6×SSC buffer at 65-70° C. for16-20 hours; wash twice in 2×SSC buffer at room temperature for 5-20minutes each; and wash twice in 1×SSC buffer at 55-70° C. for 30 minuteseach.

Non-stringent control condition (sequences that share at least 50%sequence identity will hybridize): Hybridization in 6×SSC buffer at roomtemperature to 55° C. for 16-20 hours; wash at least twice in 2×-3×SSCbuffer at room temperature to 55° C. for 20-30 minutes each.

As used herein, the term “substantially homologous” or “substantialhomology,” with regard to a polynucleotide, refers to polynucleotidesthat hybridize under stringent conditions to the reference nucleic acidsequence. For example, polynucleotides that are substantially homologousto a reference DNA coding sequence are those polynucleotides thathybridize under stringent conditions (e.g., the Moderate Stringencyconditions set forth, supra) to the reference DNA coding sequence.Substantially homologous sequences may have at least 80% sequenceidentity. For example, substantially homologous sequences may have fromabout 80% to 100% sequence identity, such as about 81%; about 82%; about83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%;about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; andabout 100%. The property of substantial homology is closely related tospecific hybridization. For example, a nucleic acid molecule isspecifically hybridizable when there is a sufficient degree ofcomplementarity to avoid non-specific binding of the nucleic acid tonon-target sequences under conditions where specific binding is desired,for example, under stringent hybridization conditions.

As used herein, the term “ortholog” refers to a gene in two or morespecies that has evolved from a common ancestral nucleotide sequence,and may retain the same function in the two or more species.

DNA has two antiparallel strands, a 5′-»3′ strand, referred to as theplus strand, and a 3′-»5′ strand, referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′-»3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, an RNA transcript will have a sequencecomplementary to the minus strand, and identical to the plus strand(except that U is substituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a DNAtarget. An antisense RNA is a molecule of RNA complementary to a sense(encoding) nucleic acid molecule.

As used herein, two nucleic acid sequence molecules are said to exhibit“complete complementarity” when every nucleotide of a sequence read inthe 5′ to 3′ direction is complementary to every nucleotide of the othersequence when read in the 3′ to 5′ direction. A nucleotide sequence thatis complementary to a reference nucleotide sequence will exhibit asequence identical to the reverse complement sequence of the referencenucleotide sequence. These terms and descriptions are well defined inthe art and are easily understood by those of ordinary skill in the art.

As used herein, the term “substantially identical” may refer tonucleotide sequences that are more than 85% identical. For example, asubstantially identical nucleotide sequence may be at least 85.5%; atleast 86%; at least 87%; at least 88%; at least 89%; at least 90%; atleast 91%; at least 92%; at least 93%; at least 94%; at least 95%; atleast 96%; at least 97%; at least 98%; at least 99%; or at least 99.5%identical to the reference sequence.

Operably linked: A first nucleotide sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence is ina functional relationship with the second nucleic acid sequence. Whenrecombinantly produced, operably linked nucleic acid sequences aregenerally contiguous, and, where necessary to join two protein-codingregions, in the same reading frame (e.g., in a polycistronic ORF).However, nucleic acids need not be contiguous to be operably linked.

The term, “operably linked,” when used in reference to a regulatorysequence and a coding sequence, means that the regulatory sequenceaffects the expression of the linked coding sequence. “Regulatorysequences,” or “control elements,” refer to nucleotide sequences thatinfluence the timing and level/amount of transcription, RNA processingor stability, or translation of the associated coding sequence.Regulatory sequences may include promoters; translation leadersequences; introns; enhancers; stem-loop structures; repressor bindingsequences; termination sequences; polyadenylation recognition sequences;etc. Particular regulatory sequences may be located upstream and/ordownstream of a coding sequence operably linked thereto. Also,particular regulatory sequences operably linked to a coding sequence maybe located on the associated complementary strand of a double-strandednucleic acid molecule.

Promoter: As used herein, the term “promoter” refers to a region of DNAthat may be upstream from the start of transcription, and that may beinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A promoter may be operably linked to a codingsequence for expression in a cell, or a promoter may be operably linkedto a nucleotide sequence encoding a signal sequence which may beoperably linked to a coding sequence for expression in a cell.

Some embodiments herein include a “plant promoter.” A plant promoter isa promoter that is capable of initiating transcription in a plant cell.

Some embodiments herein include a “tissue-preferred promoter.” Atissue-preferred promoter is a promoter that is capable of initiatingtranscription under developmental control, and include, for example andwithout limitation: promoters that preferentially initiate transcriptionin leaves, pollen, tassels, roots, seeds, fibers, xylem vessels,tracheids, and sclerenchyma. Promoters that initiate transcriptionessentially only in certain tissues are referred to as“tissue-specific.” A “cell type-specific” promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter may be apromoter which may be under environmental control. Examples ofenvironmental conditions that may initiate transcription by induciblepromoters include anaerobic conditions and the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters.

Any inducible promoter may be used in some embodiments of the invention.See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With an induciblepromoter, the rate of transcription increases in response to an inducingagent. Exemplary inducible promoters include, but are not limited to:Promoters from the ACEI system that responds to copper; In2 gene frommaize that responds to benzenesulfonamide herbicide safeners; Tetrepressor from Tn10; and the inducible promoter from a steroid hormonegene, the transcriptional activity of which may be induced by aglucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci.USA 88:10421-5).

In contrast to non-constitutive promoters, a “constitutive” promoter isa promoter that is active under most environmental conditions. Exemplaryconstitutive promoters include, but are not limited to: promoters fromplant viruses, such as the 35S promoter from CaMV; promoters from riceactin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter;and the ALS promoter, Xbal/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xbal/NcoIfragment) (PCT International Patent Publication No. WO 96/30530).

Additionally, any tissue-specific or tissue-preferred promoter may beutilized in some embodiments of the invention. Plants transformed with anucleic acid molecule comprising a coding sequence operably linked to atissue-specific promoter may produce the product of the coding sequenceexclusively, or preferentially, in a specific tissue. Exemplarytissue-specific or tissue-preferred promoters include, but are notlimited to: a root-preferred promoter, such as that from the phaseolingene; a leaf-specific and light-induced promoter such as that from cabor rubisco; an anther-specific promoter such as that from LAT52; apollen-specific promoter such as that from Zm13; and amicrospore-preferred promoter such as that from apg.

Trait or phenotype: The terms “trait” and “phenotype” are usedinterchangeably herein. For the purposes of the present disclosure,traits of particular interest include agronomically important traits, asmay be expressed, for example, in a crop plant. In some examples, atrait of particular interest is male sterility.

Transformation: As used herein, the term “transformation” refers to thetransfer of one or more nucleic acid molecule(s) into a cell. A cell is“transformed” by a nucleic acid molecule introduced into the cell whenthe nucleic acid molecule becomes stably replicated by the cell, eitherby incorporation of the nucleic acid molecule into the cellular genome,or by episomal replication. As used herein, the term “transformation”encompasses all techniques by which a nucleic acid molecule can beintroduced into such a cell. Examples include, but are not limited to:transfection with viral vectors; transformation with plasmid vectors;electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection(Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7);microinjection (Mueller et al. (1978) Cell 15:579-85);Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad.Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment(Klein et al. (1987) Nature 327:70).

Transgene: A transgene is an exogenous nucleic acid sequence. In someexamples, a transgene may be a sequence that encodes one or bothstrand(s) of a dsRNA molecule that comprises a nucleotide sequence thatis complementary to a target nucleic acid. In some examples, a transgenemay be an antisense nucleic acid sequence, the expression of whichinhibits expression of a target nucleic acid. In still other examples, atransgene may be a gene sequence (e.g., a herbicide-resistance gene), agene encoding an industrially or pharmaceutically useful compound, or agene encoding a desirable agricultural trait. In these and otherexamples, a transgene may contain regulatory sequences operably linkedto the coding sequence of the transgene (e.g., a promoter).

Vector: A vector refers to a nucleic acid molecule as introduced into acell, for example, to produce a transformed cell. A vector may includenucleic acid sequences that permit it to replicate in the host cell,such as an origin of replication. Examples of vectors include, but arenot limited to: a plasmid; cosmid; bacteriophage; and a virus thatcarries exogenous DNA into a cell. A vector may also include one or moregenes, antisense molecules, and/or selectable marker genes and othergenetic elements known in the art. A vector may transduce, transform, orinfect a cell, thereby causing the cell to express the nucleic acidmolecules and/or proteins encoded by the vector. A vector optionallyincludes materials to aid in achieving entry of the nucleic acidmolecule into the cell (e.g., a liposome, protein coating, etc.).

Unless specifically indicated or implied, the terms “a,” “an,” and “the”signify “at least one,” as used herein.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample, B. Lewin, Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and R. A.Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All temperatures are in degrees Celsius.

IV. Nucleic Acids and Systems

Plants and animals use sRNAs to direct the post-transcriptional andepigenetic regulation of target genes. Many miRNAs and theircorresponding target sequences are highly conserved. For example, targetsequences in plants that are recognized by related miRNAs in differentspecies often differ only by several nucleotides. Therefore, thecomputational prediction of target sites is possible. Jones-Rhoades andBartel (2004) Mol. Cell 14:787-99. Additionally, a functional sRNAtarget site from one plant species is likely to be functional in adifferent plant species that expresses the targeting sRNA. For example,miRNA target genes from Arabidopsis heterologously expressed inNicotiana are cleaved by endogenous Nicotiana miRNAs. Llave et al.(2002) Science 297:2053-6.

Embodiments herein include nucleic acids comprising a target site for atleast one sRNA molecule. A target site for an sRNA molecule may be, forexample, between about 20-30 nucleotides in length. For example, such atarget site may be between about 20-25 or 20-21 nucleotides in length.In particular examples, the target site may be 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, or 31 nucleotides in length. Target sites forparticular sRNAs may by identified or engineered computationally, usingprogram parameters that may be selected within the discretion of thoseskilled in the art. Target sites may be identified or engineered to bespecifically targeted by a single sRNA, or to be targeted by a group ofrelated sRNAs.

Particular embodiments include nucleic acids, comprising, for example, agene or coding region of interest that comprises an sRNA target site(e.g., an internal target site that is located within a transcribedportion of the gene or coding region), such that co-expression in a cellof the gene or coding region and the targeting sRNA results in reducedor essentially eliminated expression of the gene or coding region. Suchnucleic acids may be, for example and without limitation, a heterologousor exogenous nucleic acid in the genome of a cell (e.g., a plant cell),or a vector.

In some embodiments, a nucleic acid comprising a gene or coding regionof interest with an sRNA target site further comprises one or moreregulatory sequences that are operably linked to the gene or codingregion of interest, so as to effect the transcription of the gene orcoding region of interest in a cell; i.e., an expression construct. Inexamples, the cell is a plant cell. In particular embodiments, the geneor coding region of interest is operably linked to a constitutive plantpromoter in the expression construct. By, for example, transforming aplant cell or tissue with such an expression construct and regeneratinga plant from the plant cell or tissue, a transgenic plant may beproduced, wherein the gene or coding region of interest is transcribedin every cell or essentially every cell of the plant.

In embodiments, the gene or coding region of interest may be anagronomic gene or nucleotide sequence encoding a polypeptide ofinterest, and may also and/or alternatively include, for example andwithout limitation: genes that confer resistance to an herbicide, suchas an herbicide that inhibits the growing point or meristem, forexample, an imidazolinone or a sulfonylurea (exemplary genes in thiscategory encode mutant ALS and AHAS enzymes, as described, for example,by Lee et al. (1988) EMBO J. 7:1241, and Mild et al. (1990) Theor. Appl.Genet. 80:449, respectively); glyphosate resistance as conferred by,e.g., mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes); aroA genes and glyphosateacetyl transferase (GAT) genes, respectively); other phosphonocompounds, such as glufosinate phosphinothricin acetyl transferase (PAT)genes from Streptomyces species, including Streptomyces hygroscopicusand Streptomyces viridichromogenes); and pyridinoxy or phenoxyproprionic acids and cyclohexones (ACCase inhibitor-encoding genes).See, e.g., U.S. Pat. Nos. 4,940,835 and 6,248,876 (nucleotide sequencesof forms of EPSPs which can confer glyphosate resistance to a plant). ADNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256. See also U.S. Pat. No. 4,769,061 (nucleotidesequence of a mutant aroA gene). European patent application No. 0 333033 and U.S. Pat. No. 4,975,374 disclose nucleotide sequences ofglutamine synthetase genes, which may confer resistance to herbicidessuch as L-phosphinothricin. Nucleotide sequences of exemplary PAT genesare provided in European application No. 0 242 246, and DeGreef et al.(1989) Bio/Technology 7:61 (production of transgenic plants that expresschimeric bar genes coding for PAT activity). Exemplary of genesconferring resistance to phenoxy proprionic acids and cyclohexones, suchas sethoxydim and haloxyfop, include the Accl-S1, Accl-S2 and Accl-S3genes described by Marshall et al. (1992) Theor. Appl. Genet. 83:435.GAT genes capable of conferring glyphosate resistance are described, forexample, in WO 2005012515. Genes conferring resistance to 2,4-D,phenoxyproprionic acid and pyridyloxy auxin herbicides are described,for example, in WO 2005107437.

Nucleic acids comprising an agronomic gene or nucleotide sequenceencoding a polypeptide of interest may also include, for example andwithout limitation: a gene conferring resistance to an herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) or abenzonitrile (nitrilase gene). See, e.g., Przibila et al. (1991) PlantCell 3:169 (transformation of Chlamydomonas with plasmids encodingmutant psbA genes). Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648, and DNA molecules containing thesegenes are available under ATCC Accession Nos. 53435; 67441; and 67442.See also Hayes et al. (1992) Biochem. J. 285:173 (cloning and expressionof DNA coding for a glutathione S-transferase).

Embodiments herein also or alternatively include a set of nucleic acids,wherein the set includes at least one nucleic acid comprising a targetsite for at least one sRNA molecule. In some embodiments, the set ofnucleic acids further includes at least one nucleic acid encoding atleast one sRNA molecule targeting the nucleic acid comprising the targetsite therefore. In particular embodiments, a plant cell may betransformed (either in one step or in multiple steps) with the nucleicacids of the set. In examples, when a transgenic plant tissue or plantis regenerated from the transformed plant cell, the nucleic acidcomprising the target site for the at least one sRNA molecule isexpressed essentially in all cells other than those wherein the sRNAmolecule is expressed. Thus, when the set of nucleic acids includes anucleic acid encoding the sRNA molecule under the control of atissue-specific promoter, the nucleic acid comprising the target sitefor the at least one sRNA molecule is expressed essentially in all cellsother than cells of the tissue wherein the promoter directs expressionof the sRNA molecule.

V. Methods

In some embodiments herein, a plant cell, plant part, and/or plant maybe genetically modified to comprise at least one nucleic acid comprisinga target site for at least one sRNA molecule by any of several methodsof introducing a heterologous molecule known in the art, therebyproducing a non-natural transgenic plant cell, plant part, or plant. Inparticular embodiments herein, a heterologous molecule is introducedinto a plant cell, plant part, and/or plant by a method selected from,for example and without limitation: transformation and selectivebreeding (e.g., backcross breeding).

In some embodiments, the nucleic acid is selected such that the targetsite is a target of an endogenous sRNA of the plant wherein theheterologous nucleic acid is introduced. In particular embodiments, thetarget site is the target site of an endogenous sRNA of the plant thatis endogenously expressed in the plant in a tissue-preferred ortissue-specific manner.

Depending on the particular target gene and the level of production ofthe sRNA, embodiments herein may provide partial or complete loss ofexpression, or function, of the target gene. The inhibition in targetgene expression in different embodiments is at least a 5%, at least a10%, at least a 20%, at least a 30%, at least a 50%, at least a 75%, atleast an 80%, at least an 85%, at least a 90%, at least a 95%, or a 100%inhibition in target gene expression. Any plant species or plant cellmay be genetically modified to comprise a heterologous nucleic acidherein. In some embodiments, the plant cell that is so geneticallymodified is capable of regeneration to produce a plant. In someembodiments, plant cells that are genetically modified (e.g., host plantcells) include cells from, for example and without limitation, a higherplant, a dicotyledonous plant, a monocotyledonous plants, a consumableplant, a crop plant, and a plant utilized for its oils (e.g., an oilseedplant). Such plants include, for example and without limitation:alfalfa; soybean; cotton; rapeseed (canola); linseed; corn; rice;brachiaria; wheat; safflower; sorghum; sugarbeet; sunflower; tobacco;and grasses (e.g., turf grass). In particular examples, a geneticallymodified plant cell or plant herein includes, for example and withoutlimitation: Brassica napus; Indian mustard (Brassica juncea); Ethiopianmustard (Brassica carinata); turnip (Brassica raga); cabbage (Brassicaoleracea); Glycine max; Linum usitatissimum; Zea mays; Carthamustinctorius; Helianthus annuus; Nicotiana tabacum; Arabidopsis thaliana,Brazil nut (Betholettia excelsa); castor bean (Ricinus communis);coconut (Cocus nucifera); coriander (Coriandrum sativum); Gossypiumspp.; groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); oilpalm (Elaeis guineeis); olive (Olea eurpaea); Oryza sativa; squash(Cucurbita maxima); barley (Hordeum vulgare); sugarcane (Saccharumofficinarum); Triticum spp. (including Triticum durum and Triticumaestivum); and duckweed (Lemnaceae sp.). In some embodiments, the plantmay have a particular genetic background, as for elite cultivars,wild-type cultivars, and commercially distinguishable varieties.

According to methods known in the art, nucleic acids can be introducedinto essentially any plant. Embodiments herein may employ any of themany methods for the transformation of plants (and production ofgenetically modified plants) that are known in the art. Such methodsinclude, for example and without limitation, biological and physicaltransformation protocols for dicotyledenous plants, as well asmonocotyledenous plants. See, e.g., Goto-Fumiyuki et al. (1999) Nat.Biotechnol. 17:282-6; Miki et al. (1993) Methods in Plant MolecularBiology and Biotechnology (B. R. Glick and J. E. Thompson, Eds.), CRCPress, Inc., Boca Raton, Fla., pp. 67-88. In addition, vectors and invitro culture methods for plant cell and tissue transformation andregeneration of plants are described, for example, in Gruber and Crosby(1993) Methods in Plant Molecular Biology and Biotechnology, supra, atpp. 89-119.

Plant transformation techniques available for introducing a nucleic acidinto a plant host cell include, for example and without limitation:transformation with disarmed T-DNA using Agrobacterium tumefaciens or A.rhizogenes as the transformation agent; calcium phosphate transfection;polybrene transformation; protoplast fusion; electroporation (D'Halluinet al. (1992) Plant Cell 4:1495-505); ultrasonic methods (e.g.,sonoporation); liposome transformation; microinjection; contact withnaked DNA; contact with plasmid vectors; contact with viral vectors;biolistics (e.g., DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-3) and microparticle bombardment (Sanford et al.(1987) Part. Sci. Technol. 5:27; Sanford (1988) Trends Biotech. 6:299,Sanford (1990) Physiol. Plant 79:206; and Klein et al. (1992)Biotechnology 10:268); silicon carbide WHISKERS-mediated transformation(Kaeppler et al. (1990) Plant Cell Rep. 9:415-8); nanoparticletransformation (see, e.g., U.S. Patent Publication No.US2009/0104700A1); aerosol beaming; and polyethylene glycol(PEG)-mediated uptake. In specific examples, a heterologous nucleic acidmay be introduced directly into the genomic DNA of a plant cell.

A widely utilized method for introducing an expression vector into aplant is based on the natural transformation system of Agrobacterium.Horsch et al. (1985) Science 227:1229. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria known to be useful to geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. Kado (1991) Crit. Rev. Plant. Sci. 10:1.Details regarding Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are also available in, for example,Gruber et al., supra, Miki et al., supra, Moloney et al. (1989) PlantCell Reports 8:238, and U.S. Pat. Nos. 4,940,838 and 5,464,763.

If Agrobacterium is used for the transformation, the DNA to be insertedtypically is cloned into special plasmids; either into an intermediatevector or a binary vector. Intermediate vectors cannot replicatethemselves in Agrobacterium. The intermediate vector may be transferredinto A. tumefaciens by means of a helper plasmid (conjugation). TheJapan Tobacco Superbinary system is an example of such a system(reviewed by Komari et al. (2006) Methods in Molecular Biology (K. Wang,ed.) No. 343; Agrobacterium Protocols, 2^(nd) Edition, Vol. 1, HumanaPress Inc., Totowa, N.J., pp. 15-41; and Komori et al. (2007) PlantPhysiol. 145:1155-60). Binary vectors can replicate themselves both inE. coli and in Agrobacterium. Binary vectors comprise a selection markergene and a linker or polylinker which are framed by the right and leftT-DNA border regions. They can be transformed directly intoAgrobacterium (Holsters, 1978). The Agrobacterium comprises a plasmidcarrying a vir region. The Ti or Ri plasmid also comprises the virregion necessary for the transfer of the T-DNA. The vir region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be contained.

The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of a T-strand containing the construct and adjacentmarker into the plant cell DNA when the cell is infected by the bacteriausing a binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-21) orthe co-cultivation procedure (Horsch et al. (1985) Science 227:1229-31).Generally, the Agrobacterium transformation system is used to engineerdicotyledonous plants. Bevan et al. (1982) Ann. Rev. Genet. 16:357-84;Rogers et al. (1986) Methods Enzymol. 118:627-41. The Agrobacteriumtransformation system may also be used to transform, as well astransfer, nucleic acids to monocotyledonous plants and plant cells. SeeU.S. Pat. No. 5,591,616; Hernalsteen et al. (1984) EMBO J. 3:3039-41;Hooykass-Van Slogteren et al. (1984) Nature 311:763-4; Grimsley et al.(1987) Nature 325:1677-9; Boulton et al. (1989) Plant Mot. Biol.12:31-40; and Gould et al. (1991) Plant Physiol. 95:426-34.

The genetic manipulations of a recombinant host herein may be performedusing standard genetic techniques and screening, and may be carried outin any host cell that is suitable to genetic manipulation. In someembodiments, a recombinant host cell may be any organism ormicroorganism host suitable for genetic modification and/or recombinantgene expression. In some embodiments, a recombinant host may be a plant.Standard recombinant DNA and molecular cloning techniques used here arewell-known in the art and are described in, for example and withoutlimitation: Sambrook et al. (1989), supra; Silhavy et al. (1984)Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and Ausubel et al. (1987) Current Protocols inMolecular Biology, Greene Publishing Assoc. and Wiley-Interscience, NewYork, N.Y.

Following the introduction of a nucleic acid into a plant cell, theplant cell may be grown, and upon emergence of differentiating tissuesuch as shoots and roots, mature plants can be generated. In someembodiments, a plurality of plants can be generated. Methodologies forregenerating plants are known to those of ordinary skill in the art andcan be found, for example, in: Plant Cell and Tissue Culture (Vasil andThorpe, Eds.), Kluwer Academic Publishers, 1994. Genetically modifiedplants described herein may be cultured in a fermentation medium orgrown in a suitable medium such as soil. In some embodiments, a suitablegrowth medium for higher plants may be any growth medium for plants,including, for example and without limitation; soil, sand, any otherparticulate media that support root growth (e.g., vermiculite, perlite,etc.) or hydroponic culture, as well as suitable light, water andnutritional supplements that facilitate the growth of the higher plant.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype, and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker that has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) “Protoplasts Isolationand Culture,” in Handbook of Plant Cell Culture, Macmillian PublishingCompany, New York, pp. 124-176; and Binding (1985) Regeneration ofPlants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73.Regeneration can also be performed from plant callus, explants, organs,pollens, embryos or parts thereof. Such regeneration techniques aredescribed generally in Klee et al. (1987) Ann. Rev. Plant Phys.38:467-86.

In other embodiments, the plant cells which are transformed are notcapable of regeneration to produce a plant. Such cells may be employed,for example, in developing a plant cell line having a relevantphenotype, for example, herbicide resistance and/or male sterility.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells can also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, or gfp genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

A transgenic plant containing a heterologous molecule herein can beproduced through selective breeding, for example, by sexually crossing afirst parental plant comprising the molecule, and a second parentalplant, thereby producing a plurality of first progeny plants. A firstprogeny plant may then be selected that is resistant to a selectablemarker (e.g., glyphosate, resistance to which may be conferred upon theprogeny plant by the heterologous molecule herein). The first progenyplant may then by selfed, thereby producing a plurality of secondprogeny plants. Then, a second progeny plant may be selected that isresistant to the selectable marker. These steps can further include theback-crossing of the first progeny plant or the second progeny plant tothe second parental plant or a third parental plant.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Otherbreeding methods commonly used for different traits and crops are knownin the art. Backcross breeding has been used to transfer genes for asimply inherited, highly heritable trait into a desirable homozygouscultivar or inbred line, which is the recurrent parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.After the initial cross, individuals possessing the phenotype of thedonor parent are selected and repeatedly crossed (backcrossed) to therecurrent parent. The resulting parent is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent.

A nucleic acid may also be introduced into a predetermined area of theplant genome through homologous recombination. Methods to stablyintegrate a polynucleotide sequence within a specific chromosomal siteof a plant cell via homologous recombination have been described withinthe art. For instance, site specific integration as described in USPatent Application Publication No. 2009/0111188 A1 involves the use ofrecombinases or integrases to mediate the introduction of a donorpolynucleotide sequence into a chromosomal target. In addition, PCTInternational Patent Publication No. WO 2008/021207 describes zincfinger mediated-homologous recombination to stably integrate one or moredonor polynucleotide sequences within specific locations of the genome.The use of recombinases such as FLP/FRT as described in U.S. Pat. No.6,720,475, or CRE/LOX as described in U.S. Pat. No. 5,658,772, can beutilized to stably integrate a polynucleotide sequence into a specificchromosomal site. Finally, the use of meganucleases for targeting donorpolynucleotides into a specific chromosomal location is described inPuchta et al. (1996) Proc. Natl. Acad. Sci. USA 93:5055-60.

Other various methods for site specific integration within plant cellsare generally known and applicable. Kumar et al. (2001) Trends PlantSci. 6(4):155-9. Furthermore, site-specific recombination systems thathave been identified in several prokaryotic and lower eukaryoticorganisms may be applied for use in plants. Examples of such systemsinclude, but are not limited too; the R/RS recombinase system from thepSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki et al.(1985)J. Mol. Biol. 182:191-203), and the Gin/gix system of phage Mu(Maeser and Kahlmann (1991)Mol. Gen. Genet. 230:170-6).

Various assays can be employed in connection with the nucleic acidmolecule of certain embodiments herein. In addition to phenotypicobservations, the following techniques are useful in detecting thepresence of a nucleic acid molecule in a plant cell. For example, thepresence of the molecule can be determined by using a primer or probe ofthe sequence, an ELISA assay to detect an encoded protein, a Westernblot to detect the protein, or a Northern or Southern blot to detect RNAor DNA. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of a recombinant construct in specific plant organs andtissues.

Southern analysis is a commonly used detection method, wherein DNA iscut with restriction endonucleases and fractionated on an agarose gel toseparate the DNA by molecular weight and then transferring to nylonmembranes. It is then hybridized with the probe fragment which wasradioactively labeled with ³²P (or other probe labels) and washed in anSDS solution.

Likewise, Northern analysis deploys a similar protocol, wherein RNA iscut with restriction endonucleases and fractionated on an agarose gel toseparate the RNA by molecular weight and then transferring to nylonmembranes. It is then hybridized with the probe fragment which wasradioactively labeled with ³²P (or other probe labels) and washed in anSDS solution. Analysis of the RNA (e.g., mRNA) isolated from the tissuesof interest can indicate relative expression levels. Typically, if themRNA is present or the amount of mRNA has increased, it can be assumedthat the corresponding transgene is being expressed. Northern analysis,or other mRNA analytical protocols, can be used to determine expressionlevels of an introduced transgene or native gene.

Nucleic acids herein, or segments thereof, may be used to design primersfor PCR amplification. In performing PCR amplification, a certain degreeof mismatch can be tolerated between primer and template. Mutations,insertions, and deletions can be produced in a given primer by methodsknown to an ordinarily skilled artisan.

Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies,Foster City, Calif.), is another method of detecting and quantifying thepresence of a DNA sequence. Briefly, a FRET oligonucleotide probe isdesigned with one oligo within the transgene and one in the flankinggenomic sequence for event-specific detection. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization.

VI. Plants

Some embodiments herein provide transgenic plants comprising at leastone nucleic acid comprising a target site for at least one sRNAmolecule, such as may be regenerated from stably transformed plant cellsor tissues, or may be produced by introgression of such a nucleic acidfrom a donor line. Such plants may be used or cultivated in any manner,wherein presence of the transforming polynucleotide(s) of interest isdesirable. Accordingly, transgenic plants may be engineered to, interalia, have one or more desired traits (e.g., male sterility), bytransformation, and then may be cropped and cultivated by any methodknown to those of skill in the art. Particular embodiments hereinprovide parts, cells, and/or tissues of such transgenic plants. Plantparts, without limitation, include seed, endosperm, ovule and pollen. Insome embodiments, the plant part is a seed.

Representative, non-limiting example plants include Arabidopsis; fieldcrops (e.g., alfalfa, barley, bean, clover, corn, cotton, flax, lentils,maize, pea, rape/canola, rice, rye, safflower, sorghum, soybean,sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet,Brassica, broccoli, Brussels sprouts, cabbage, carrot, cauliflower,celery, cucumber (cucurbits), eggplant, lettuce, mustard, onion, pepper,potato, pumpkin, radish, spinach, squash, taro, tomato, and zucchini);fruit and nut crops (e.g., almond, apple, apricot, banana, blackberry,blueberry, cacao, cassava, cherry, citrus, coconut, cranberry, date,hazelnut, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon,nectarine, orange, papaya, passion fruit, peach, peanut, pear,pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut,and watermelon); tree woods and ornamentals (e.g., alder, ash, aspen,azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm, fir,ivy, jasmine, juniper, oak, palm, poplar, pine, redwood, rhododendron,rose, and rubber).

To confirm the presence of a transforming polynucleotide(s) of interestin a regenerating plant, a variety of assays may be performed. Suchassays include, for example and without limitation: biochemical assays,such as detecting the presence of a protein product, e.g., byimmunological means (ELISA and/or Western blots) or by enzymaticfunction; plant part assays (e.g., leaf or root assays); and analysis ofthe phenotype of the plant.

There are numerous steps in the development of any novel, desirableplant germplasm, which may begin with the generation of a transgeniccrop plant. In some embodiments, a transgenic plant comprising at leastone nucleic acid comprising a target site for at least one sRNA molecule(e.g., a male sterile plant) may be used in a plant breeding and/orgermplasm development program.

Plant breeding begins with the analysis and definition of problems andweaknesses of the current germplasm, the establishment of program goals,and the definition of specific breeding objectives. The next step isselection of germplasm that possess the traits to meet the programgoals. The goal is to combine in a single variety an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include higher seed yield, resistance to diseasesand insects, better stems and roots, tolerance to drought and heat, andbetter agronomic quality.

The choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods include pedigree selection, modifiedpedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In embodiments herein, a transgene comprising a target site for at leastone sRNA molecule may be introduced into a plant germplasm, for example,to develop novel inbred lines that are characterized by thetissue-specific expression of the transgene, under the control of thesRNA molecule. A particular advantage of such a development program maybe that the generality of the RNAi pathway results in a higherpenetrance of the transgenic phenotype than would otherwise beattainable, for example, by other control mechanisms. In certainembodiments, a herbicide tolerance gene that comprises a target site foran sRNA molecule is introduced into a plant germplasm, such that thetrait of male sterility is stably inherited and accomplishes hybridproduction with a minimal expenditure of valuable resources.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1: Tissue-Specific miRNAs

Many plant miRNAs have a distinct developmental and tissue-specificexpression pattern. For example, Arabidopsis thaliana miR171 (previouslyknown as miR39) accumulates predominantly in inflorescence tissues.miR171 is produced from an intergenic region in chromosome III andfunctionally interacts with mRNA targets encoding several members of theScarecrow-like (SCL) family of putative transcription factors. Llave etal. (2002) Science 297:2053-6. The interaction results intissue-specific cleavage of target mRNAs within the region ofcomplementarity between miR171 and a native gene mRNA. Transgene mRNAscarrying the target site sequence are also recognized and cleaved.

Similarly, Zea mays (maize) miR156 (SEQ ID NO:1) and miR529 (SEQ IDNO:2) are expressed in a developmental and tissue-specific manner. Chucket al. (2010) Development 137:1243-50; Zhang et al. (2009) PloS GeneticsNovember 5. miR156 is temporally regulated, being expressed mainlyduring the early vegetative stage, but barely detectable during thereproductive stage. In contrast, miR529 is not detected during thevegetative stage, but can be detected in roots and during thereproductive stage in reproductive tissues. Zhang et al. (2009), supra.miR156 and miR529 share a 14 to 16 nucleotide homology, and both targetthe tasselsheath4 (referred to herein as tsh4) mRNA. tsh4 regulatesbract development and the establishment of meristem boundaries in maize.Chuck et. al. (2010), supra.

miRNA Microarray in Various Maize Tissues:

Tissues from maize inbred line B104 were used for miRNA profiling. Leaftissues (at V2, V8 and flag leaf stages); immature tassel tissues (0.5to 2.5 cm length); mature tassel tissues (tassel visible in the whorl);immature ear tissues (0.5 to 2 cm length); and mature ear tissues(visible ear shoot) were collected and immediately submerged in liquidnitrogen and stored at −80° C. Total RNA and low molecular weight RNAswere extracted using a mirVana™ miRNA isolation kit (INVITROGEN;Carlsbad, Calif.) following the manufacturer's instructions. RNA qualityand quantity were assessed by optical density with a NANODROP 2000spectrophotometer (THERMO SCIENTIFIC; Wilmington, Del.) and by gelelectrophoresis. miRNA microarray was performed by a commercial provider(LC SCIENCES; Houston, Tex.) using μParaflo® microfluidicoligonucleotide microarray technology. Zhou et al., (2012) Methods Mol.Biol. 822:153-82. All unique plant miRNA sequences available in MiRBase™(mirbase.org) were printed on the arrays and used for miRNA profiling.

Data regarding miRNA expression of miR156, miR529, and miR319 (SEQ IDNO:3) are shown in Table 1. The data demonstrate that miR529 isexpressed at high levels in the immature tassel, with medium expressionin the mature ear and mature tassel, and very low expression in leavesand immature ear, whereas miR156 is expressed at high or medium levelsin all tissues examined except the immature ear and immature tassel.Given the preferential expression of miR529 in tassel and ear tissues,an miR529 recognition site was selected to exemplify tissue-specificengineering.

TABLE 1 miRNA microarray expression profiling of miR529, miR156, andmiR319 in maize tissues. Numbers represent relative expression levels.Tissue miR529 miR156 miR319 Leaf V2 199 4648 13 Leaf V8 34 1337 8 FlagLeaf 30 1657 16 Immature Ear 90 112 3905 Immature Tassel 3174 78 2078Mature Ear 1405 1595 4616 Mature Tassel 1512 2810 1566

Example 2: Plant Transformation Vectors

In order to investigate whether endogenous sRNA molecules can be used toconfer male sterility, the following nucleic acid constructs wereengineered and produced.

Transient Transformation Constructs: pDAB112375 (ZmUbi1v2/AAD1 v3/nativeWT miR529-156 site/ZmPer5 3′UTR); pDAB112376 (ZmUbi1v2/AAD1 v3/WT miR529target site with miR156 site mutated/ZmPer5 3′UTR); pDAB112377(ZmUbi1v2/AAD1 v3/miR529 perfect target site with miR156 sitemutated/ZmPer5 3′UTR); and pDAB113016 (ZmUbi1v2/AAD1 v3/ZmPer5 3′UTR)(non miRNA target control).

Stable Transformation Constructs

(All constructs contain ZmPer5 3′UTR/OsAct1 v2/PATv9/Zm Lip 3′UTR withinT-DNA): pDAB113018 (ZmUbi1v2/AAD1 v3/native WT miR529-156 site/ZmPer53′UTR); pDAB113019 (ZmUbi1v2/AAD1 v3/WT miR529 target site with miR156site mutated/ZmPer5 3′UTR); pDAB113020 (ZmUbi1v2/AAD1 v3/miR529 perfecttarget site with miR156 site mutated/ZmPer5 3′UTR); and pDAB113021(ZmUbi1v2/AAD1 v3/ZmPer5 3′UTR) (non miRNA target control).

miRNA Target Sites:

The miRNA target site sequences for miR156 and miR529 were obtained fromMiRBase™. A native target site within a corn tsh4 gene (Chuck et al.(2010), supra) was used to design target sites for this disclosure. Thetsh4 target site designed (SEQ ID NO:4) contains overlapping miR156 andmiR529 binding sites at the 3′ end of the coding region. Table 2A. Themajority of tsh4 transcript cleavage (68%) occurs between base pairs 10and 11 of the predicted microRNA binding site for miR529 (underlined CTbases in the tsh4 sequence in Table 2A, 2B and 2C), whereas 31% occursnear the predicted cleavage site for miR156 (between base pairs 5 and 7of miR529). Chuck et al. (2010), supra. The native tsh4 target sitesequence was engineered, such that the miR156 binding site within thetsh4 sequence was mutated without interfering with the miR529 bindingand cleavage site. Table 2A shows the native tsh4 target site withbinding sites specific to miR156 or mR529, which was used in theconstruction of pDAB112375. In Tables 2A, 2B, and 2C, mismatches areshown as a “star” (*) while a dot (⋅) indicates a “G-U” wobble. Table 2Band Table 2C illustrate the modifications (lower case) made in the tsh4target site as used in constructs of pDAB112376 and pDAB112377,respectively.

TABLE 2Sequences of native tsh4 binding site and mutated versions used in maizetransformations; pDAB112375 (Table 2A), pDAB112376 (Table 2B), and pDAB112377 (Table 2C).Mismatches are shown as a “star” (*) while a dot (●) indicates a “G-U”wobble. A. Sequence Alignments 5′ GACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCA 3′native tsh4 site (SEQ ID NO: 4)              ||||||*|||||||||||||          3′ CACGAGUGAGAGAAGACAGU 5′ miR156 (SEQ ID NO: 1)        **|||||●|||||||||||||      3′ UCCGACAUGAGAGAGAGAAGA 5′miR529 (SEQ ID NO: 2)Native tsh4 sequence showing homology to miR156 and miR529 micro-RNAs. miR529-directedcleavage site CT is underlined. B. Sequence Alignments 5′GACTCagGCTGTaCTCTCTtaCTTCacaaagtACTCA 3′ mutant tsh4 (SEQ ID NO: 7)             ||*|||*||**||||****|           3′ CACGAGUGAGAGAAGACAGU 5′miR156 (SEQ ID NO: 1)              |||||||||**||||*      3′UCCGACAUGAGAGAGAGAAGA 5′ miR529 (SEQ ID NO: 2)miR529 homology to tsh4 target sequence improved, preserving the cleavage site, with miR156binding site mutated. C. Sequence Alignments 5′GACTCagGCTGTaCTCTCTctCTTCacaaagtACTCA 3′ mutant tsh4 (SEQ ID NO: 8)             ||*|||*||||||||****|           3′ CACGAGUGAGAGAAGACAGU 5′miR156 (SEQ ID NO: 1)              |||||||||**||||*      3′UCCGACAUGAGAGAGAGAAGA 5′ miR529 (SEQ ID NO: 2)miR529 homology to tsh4 target sequence further improved, preserving the cleavage site, withmiR156 binding site mutated.

GATEWAY® (INVITROGEN) entry vectors were constructed by standardmolecular cloning methods, and were used as expression vectors fortransient mRNA expression in maize cells. A starting vector expressioncassette (pDAB113016) comprised a sequence (SEQ ID NO:5) that includedan AAD1 v3 coding region followed by a fragment comprising a 3′untranslated region (UTR) from a maize peroxidase 5 gene (ZmPer5 3′UTRv2; U.S. Pat. No. 6,699,984). AAD1 is a herbicide tolerance gene thatencodes aryloxyalknoate dioxygenase (U.S. Pat. No. 7,838,733; Wright etal. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:20240-5), and thus conferstolerance to herbicidal compounds such as Haloxyfop and Quizalofop.Expression of this coding region in plant cells transformed with DNA ofplasmid pDAB113016 (and derivative plasmids below) is under the controlof a copy of a maize ubiquitin 1 promoter with associated intron1 (U.S.Pat. No. 5,510,474) and a fragment comprising a 3′UTR from a maizeperoxidase 5 gene, as above.

Other entry vector/plant transformation vectors were constructed asderivatives of pDAB113016. For example, pDAB112375 comprises a sequence(SEQ ID NO:6) that includes the AAD1 coding region of pDAB113016,followed by a native tsh4 miR529-156 target site (SEQ ID NO:4; Table 3)positioned between the end of the AAD1 coding region and the ZmPer53′UTR. Plasmid pDAB112376 is a further derivative of pDAB113016, andcomprises a sequence (SEQ ID NO: 7) that includes the AAD1 coding regionof pDAB113016, followed by a modified tsh4 target sequence comprising anative miR529 binding site, with a mutated miR156 binding site (SEQ IDNO:8; Table 3) positioned between the end of the AAD1 coding region andthe ZmPer5 3′UTR. Further, plasmid pDAB112377 is a derivative ofpDAB113016, and comprises a sequence (SEQ ID NO:9) that includes theAAD1 coding region of pDAB113016, followed by a modified tsh4 targetsite comprising a native miR529 binding site, with a second version of amutated miR156 binding site (SEQ ID NO:10; Table 3) positioned betweenthe end of the AAD1 coding region and the ZmPer5 3′UTR.

A further plant transformation plasmid, pDAB112378, was constructed toexpress an artificial miRNA comprising the miR529 sequence (SEQ IDNO:2). A DNA fragment containing the sequence of miR529 was insertedinto a rice miR528 precursor sequence to build artificial miRNA sequenceCMSRF9973.1 (SEQ ID NO:11). Production of miRNA CMSRF9973.1 transcriptswas under the control of a copy of a maize ubiquitin 1 promoter withintron1, as above, and a fragment comprising a 3′UTR from a maizeperoxidase 5 gene, as above.

A negative control plasmid (pDAB112330) comprised a Cry34Ab1 proteincoding region (U.S. Pat. No. 8,273,535) under the expression control ofa copy of a maize ubiquitin 1 promoter with intron1, as above, and afragment comprising a 3′UTR from a maize ubiquitin 1 gene (essentiallyas in GENBANK Accession No. S94464.1).

A further negative control plasmid, pDAB100286, comprised a yellowfluorescent protein (YFP) marker gene coding region (Shagin et al.(2004) Mol. Biol. Evol. 21:841-50) under the expression control of acopy of a maize ubiquitin 1 promoter with intron1, as above, and afragment comprising a 3′UTR from a maize peroxidase 5 gene, as above.

TABLE 3 Exemplary miRNA target sites. SEQ ID NO: 4GACTCCAGCTGTGCTCTCTCTCTTCTGTCAACTCA SEQ ID NO: 7GACTCAGGCTGTACTCTCTTACTTCACAAAGTACTCA SEQ ID NO: 8GACTCAGGCTGTACTCTCTCTCTTCACAAAGTACTCA

Transformation/expression vectors for Agrobacterium-mediated maizeembryo transformation were constructed through the use of standardcloning methods and GATEWAY® recombination reactions employing a typicaldestination binary vector (pDAB101849) and entry vectors, as describedabove. Binary destination vector pDAB101849 comprises a herbicidetolerance gene (phosphinothricin acetyl transferase (PAT); Wehrmann etal. (1996) Nat. Biotechnol. 14: 1274-8) under the expression control ofa rice actin promoter with associated intron1 and 5′UTR (essentially asdisclosed as bases 12 to 1411 of GENBANK Accession No. EU155408.1). Afragment comprising a 3′UTR from a maize lipase gene (ZmLip 3′UTR, U.S.Pat. No. 7,179,902) was used to terminate transcription of the PAT mRNA.The GATEWAY® recombination reaction was used to move the ZmUbi1/AAD-1v3/ZmPer5 3′UTR expression cassette, with or without miR529 and miR156target sites, into the destination binary vector, between the T-DNAborders and upstream of the PAT expression cassette.

TABLE 4 miRNA tsh4 target content of binary vectors. Binary VectorEntry/WHISKERS Vector tsh4 Target Sequence pDAB113018 pDAB112375 SEQ IDNO: 4 pDAB113019 pDAB112376 SEQ ID NO: 7 pDAB113020 pDAB112377 SEQ IDNO: 8 pDAB113021 pDAB113016 None

In addition, transformation vector pDAB109812 was constructed throughthe use of standard GATEWAY® recombination reactions employing a typicalbinary destination vector (pDAB 101847). Binary destination vector pDAB101847 comprises an AAD1 herbicide tolerance coding region (as above)under the expression control of a sugarcane bacilliform virus (SCBV)promoter (SEQ ID NO:12); essentially as described in U.S. Pat. No.6,093,569. A synthetic 5′UTR sequence (SEQ ID NO:13), comprised ofsequences from a Maize Streak Virus (MSV) coat protein gene 5′UTR andintron 6 from a maize Alcohol Dehydrogenase 1 (ADH1) gene, is positionedbetween the 3′ end of the SCBV promoter segment and the start codon ofthe AAD1 coding region. A fragment comprising a 3′UTR from a maizelipase gene, as above, was used to terminate transcription of the AAD1mRNA. Transformation vector pDAB109812 was constructed by adding ayellow fluorescent protein marker gene cassette (Phi-YFP™ coding regioncontaining a potato intron ST-LS1 from EVROGEN, Moscow, Russia) topDAB101847 by standard molecular cloning and GATEWAY® recombinationreactions. Expression of Phi-YFP™ in vector pDAB109812 is under thecontrol of a maize ubiquitin 1 promoter with associated intron1, asabove, and a maize ZmPer5 3′UTR, as above.

The structures of all plasmids were confirmed by restriction enzymedigests and determinations of the DNA base sequences of relevant regionswere confirmed by standard molecular biological techniques.

Example 3: Transient Expression of AAD1

Zea mays embryos from inbred line B104 that express AAD1 mRNAs havingmiRNA target sites within the mRNA were produced. Control tissues havingno miRNA target sites in the AAD1 mRNA, or having no AAD1 geneexpression cassette, were also produced. Other control tissues wereproduced that had only a YFP gene expression cassette (and no AAD1construct). Preparations of plant transformation DNA moleculesconstructed as described in EXAMPLE 2 were delivered into maize B104immature embryos via particle bombardment transformation.

Plasmid DNAs of pDAB112375, pDAB112376, pDAB112377, pDAB113016,pDAB100286, pDAB112378 and pDAB112330 were isolated and purified fromEscherichia coli by standard techniques, and diluted in TE buffer (10 mMTris HCl plus 1 mM EDTA, pH 8) to 1.0 μg/μL.

Gold Particles Stock Preparation:

Gold particle stock was prepared by weighing 50 mg of 1 μm goldmicrocarriers (BIO-RAD LABORATORIES, Richmond, Calif.) into a sterile2.0 mL microcentrifuge tube, followed by 3 washes with 100% ethanol andpelleting at 1500×g for 2 minutes in a microcentrifuge. The particleswere then washed 3 times with sterile water as above. Finally, 500 μLsterile 50% glycerol was added to the gold particles in the tube and thesuspension was stored at −20° C.

Embryo Isolation and Culture:

(Day 1) Surface-sterilized B104 immature ears (10 to 12 dayspost-pollination) were used for the isolation of embryos. Excisedimmature embryos from 3 to 4 ears were pooled into 2 mL microfuge tubescontaining 2.0 mL liquid medium (LS basal medium (Linsmaier and Skoog,(1965) Physiologia Plantarum 18:100-27) containing CHU N6 vitamins (Chuet al. (1975) Scientia Sinica 18:659-68), 700 mg/L L-Proline, 68.5 gm/Lsucrose, 36 gm/L D(+) glucose, and 1.5 mg/L 2,4-D). After embryoisolation, the liquid medium was removed and discarded. Embryos wereremoved and placed onto plates (40 embryos per plate) containing asemi-solid osmotic medium (MS basal medium (Murashige and Skoog, (1962)Physiologia Plantarum 15:473-497), 2 mg/L Glycine, 500 μg/L Thiamine,500 μg/L Pyroxidine, 50 μg/L Nicotinic Acid., 120 gm/L sucrose, 100 mg/Lmyo-inositol, and 2.4 gm/L GELRITE) The embryos were arranged in a 6×7grid within the target area for particle bombardment. Three replicateplates were prepared for each construct to be tested. All plates wereincubated at 28° C. under continuous low light (50 μEm⁻² sec⁻¹) for 24hours prior to particle bombardment.

Coating Gold Particles with DNA:

(Day 2) Tubes of frozen gold particles glycerol stock were thawed on icefor a few minutes, then vortexed for 2 minutes at high speed until auniform gold suspension was produced. Gold suspension was pipetted intosterile microcentrifuge tubes (50 each), with an intermediate vortexingof the particle stock between each tube to maintain a uniformsuspension. 2.5 μg each of appropriate plasmid DNAs were included.Instantaneous mixing of experimental components was accomplished bygently vortexing the microcentrifuge tube while 2.5 appropriate DNAs (1μg/μL), 50 μL ice-cold sterile 2.5 M CaCl₂), and 20 μL of sterile 0.1 Mspermidine were added (in that order). The tubes were then vortexed athigh speed at 4° C. for 20 minutes. The gold/DNA particles were thenwashed 3 times with 100% ethanol and finally suspended in 30 μL 100%ethanol. The tubes were placed on ice and were used within 2 hrs. fromthe time of preparation.

Preparation of Macrocarriers with Gold/DNA Microcarriers:

The following operations were performed in a laminar flow hood. Sterilemacrocarriers for the BIOLISTIC PDS-1000/He PARTICLE DELIVERY SYSTEM(BIO-RAD LABORATORIES, Inc.) were placed in sterile Petri dishes. Tubesof gold/DNA microcarriers were vortexed and 5 μL gold/DNA suspension wasquickly spread onto the appropriate macrocarrier. Macrocarriers withgold/DNA were allowed to dry for 10 mins. prior to use for particlebombardment.

Particle Bombardment Transformation:

After sterilizing 1100 psi rupture disks by quickly submerging them into70% isopropanol, a rupture disk was inserted into the disc retainingcap. The macrocarriers and sterile stopping screens were loaded into thelaunch assembly and placed in the chamber. Each of the plates witharranged embryos was placed on the chamber shelf at 6 cm distance. Thechamber was then evacuated to, and held at, 28 mm Hg vacuum, and the“fire” button was held until the rupture disk burst. The vacuum was thenreleased and the target plate was covered and sealed with 3M™ Micropore™medical tape. All plates were then incubated for 24 hrs. under 50 μEm⁻²sec⁻¹ continuous light at 28° C.

On Day 3 of the experiment, embryos were removed from the plates inbatches of 40 embryos/sampling tube, and sent for protein and molecularanalyses.

Example 4: Stable Expression of AAD1

Agrobacterium-mediated transformation was used to stably integrate achimeric gene into the plant genome, and thus generate transgenic maizecells, tissues, and plants that produce AAD1 mRNAs having miRNA targetsites within the mRNA. Control tissues having no miRNA target sites inthe AAD1 mRNA were also produced. Transformed tissues were selected bytheir ability to grow on Haloxyfop- or Bialaphos-containing medium.

Agrobacterium Culture Initiation:

Glycerol stocks of the project vectors were provided in theAgrobacterium tumefaciens host strain DAt13192 (PCT International PatentPublication No. WO2012/016222A2). Agrobacterium cultures were streakedfrom glycerol stocks onto AB minimal medium (Watson, et al. (1975) J.Bacteriol. 123:255-64) and incubated at 20° C. in the dark for 3 dayscontaining appropriate antibiotics. The cultures were then streaked ontoa plate of YEP medium (g/L: yeast extract, 10; Peptone, 10; NaCl, 5)with antibiotics and incubated at 20° C. in the dark for 1 day.

On the day of an experiment, a mixture of Inoculation Medium andacetosyringone (Frame et al. (2011) Methods Mol. Biol. 710:327-41) wasprepared in a volume appropriate to the number of constructs in theexperiment and pipetted into a sterile, disposable, 250 mL flask.Inoculation Medium contains: 2.2 g/L MS salts; 1× ISU Modified MSVitamins (Frame et al. (2011), supra) 68.4 g/L sucrose; 36 g/L glucose;115 mg/L L-proline; and 100 mg/L myo-inositol; at pH 5.4).Acetosyringone was added to the flask containing Inoculation Medium to afinal concentration of 200 from a 1 M stock solution in 100% dimethylsulfoxide.

For each construct, 1 or 2 inoculating loop-fulls of Agrobacterium fromthe YEP plate were suspended in 15 mL Inoculation Medium/acetosyringonemixture inside a sterile, disposable, 50 mL centrifuge tube, and theoptical density of the solution at 550 nm (OD₅₅₀) was measured in aspectrophotometer. The suspension was then diluted to OD₅₅₀ of 0.3 to0.4 using additional Inoculation Medium/acetosyringone mixture. The tubeof Agrobacterium suspension was then placed horizontally on a platformshaker set at about 75 rpm at room temperature, and shaken for 1 to 4hours before use.

Ear Sterilization and Embryo Isolation:

Ears from Zea mays cultivar B104 were produced in a greenhouse andharvested 10 to 12 days post pollination. Harvested ears were de-huskedand surface-sterilized by immersion in a 20% solution of commercialbleach (ULTRA CLOROX® Germicidal Bleach, 6.15% sodium hypochlorite; withtwo drops of TWEEN™ 20) for 20 minutes, followed by three rinses insterile, deionized water inside a laminar flow hood. Immature zygoticembryos (1.8 to 2.2 mm long) were aseptically excised from each ear anddistributed into one or more micro-centrifuge tubes containing 2.0 mLAgrobacterium suspension into which 2 μL 10% BREAK-THRU® 5233 surfactant(EVONIK INDUSTRIES; Essen, Germany) had been added.

Agrobacterium Co-Cultivation:

Following isolation, the embryos were placed on a rocker platform for 5minutes. The contents of the tube were then poured onto a plate ofCo-cultivation Medium (4.33 g/L MS salts; 1×ISU Modified MS Vitamins; 30g/L sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH(3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid); 100mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO₃;200 acetosyringone in DMSO; and 3 g/L agar (SIGMA-ALDRICH, plant cellculture tested) at pH 5.8). The liquid Agrobacterium suspension wasremoved with a sterile, disposable, transfer pipette, and theco-cultivation plate containing the embryos was placed at the back ofthe laminar flow hood with the lid ajar for 30 minutes, after which timethe embryos were oriented with the scutellum facing up using sterileforceps with the aid of a microscope. The plate was returned to the backof the laminar flow hood with the lid ajar for a further 15 minutes. Theplate was then closed, sealed with 3M™ Micropore™ medical tape, andplaced in an incubator at 25° C. with continuous light at approximately60 μEm⁻² sec⁻¹ light intensity.

Callus Selection and Regeneration of Transgenic Events:

Following the co-cultivation period, embryos were transferred to RestingMedium (4.33 g/L MS salts; 1×ISU Modified MS Vitamins; 30 g/L sucrose;700 mg/L L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO₃; 0.5 g/L MES(2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIESLABR.; Lenexa, Kans.); 250 mg/L Cefotaxime; and 7.0 gm/L agar; at pH5.8). No more than 36 embryos were moved to each plate. The plates werewrapped with Micropore™ tape and incubated at 27° C. with continuouslight at approximately 50 μmol m⁻² s⁻¹ light intensity for 7 to 10 days.Callused embryos (<18/plate) were then transferred onto Selection MediumI, which is comprised of Resting Medium (above), but with only 6.5 g/Lagar, and with either 100 nM R-Haloxyfop acid (0.0362 mg/L; forselection of transformants harboring the AAD1 gene) or 5.0 mg/LBialaphos (for selection of transformants harboring the PAT gene), asappropriate. Bialaphos was provided as Herbiace® (20% ai). The plateswere wrapped with Micropore™ tape and incubated at 27° C. withcontinuous light at approximately 50 μEm⁻² sec⁻¹ light intensity for 7days. Callused embryos (<12/plate) were then transferred to SelectionMedium II, which is comprised of Resting Medium (above) but with only6.5 g/L agar, and with either 50 nM R-Haloxyfop acid (0.0181 mg/L) or5.0 mg/L Bialaphos, as appropriate. The plates were wrapped andincubated at 27° C. with continuous light at approximately 50 μEm⁻²sec⁻¹ light intensity for 14 days.

At this stage resistant calli (<9/plate) were moved to Pre-RegenerationMedium (4.33 g/L MS salts; 1×ISU Modified MS Vitamins; 45 g/L sucrose;350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L Casein EnzymaticHydrolysate; 1.0 mg/L AgNO₃; 0.5 g/L MES; 0.5 mg/L naphthaleneaceticacid in NaOH; 2.5 mg/L abscisic acid in ethanol; 1 mg/L6-benzylaminopurine; 250 mg/L Cefotaxime; 5.5 g/L agar; and either 50 nMR-Haloxyfop acid or 3.0 mg/L Bialaphos, as appropriate; at pH 5.8). Theplates were wrapped and incubated at 27° C. with continuous light atapproximately 50 μEm⁻² sec⁻¹ light intensity for 7 days. Regeneratingcalli (<6/plate) were then transferred to Regeneration Medium inPhytatrays™ (SIGMA-ALDRICH) and incubated at 28° C. with 16 hourslight/8 hours dark per day at approximately 150 μmol m⁻² s⁻¹ lightintensity for 14 days or until shoots developed. Regeneration Mediumcontains 4.33 g/L MS salts; 1×ISU Modified MS Vitamins; 60 g/L sucrose;0.50 g/L IVIES; 125 mg/L Cefotaxime; 5.5 g/L agar; and either 50 nMR-Haloxyfop acid or 3.0 mg/L Bialaphos, as appropriate; at pH 5.8. Smallshoots with primary roots were then isolated and transferred toElongation Medium without selection (i.e., Regeneration Medium withoutR-Haloxyfop acid or Bialaphos) for further growth. Rooted plantletsabout 6 cm or taller were transplanted into soil and moved to a growthchamber for hardening off.

Transfer and Establishment of to Plants in the Greenhouse for Assay andSeed Production:

Transformed plant tissues selected by their ability to grow on mediumcontaining either Haloxyfop or Bialaphos, as appropriate, weretransplanted from Phytatrays™ to small pots (T. O. Plastics, 3.5″ SVD)filled with growing media (PROMIX BX; Premier Tech Horticulture),covered with Humidomes™ (ARCO PLASTICS Ltd.), and then hardened-off in agrowth room (28° C. day/24° C. night, 16-hour photoperiod, 50-70% RH,200 μEm⁻² sec⁻¹ light intensity). When plants reached the V3-V4 stage,they were transplanted into SUNSHINE CUSTOM BLEND 160 soil mixture andgrown to flowering in the greenhouse (Light Exposure Type: Photo orAssimilation; High Light Limit: 1200 PAR; 16-hour day length; 27° C.day/24° C. night). Putative transgenic plantlets were analyzed fortransgene copy number by quantitative real-time PCR assays using primersdesigned to detect relative copy numbers of the transgenes, and eventsselected for advancement were transplanted into 5 gallon pots.Observations were taken periodically to track any abnormal phenotypes.

Plants of the T₁ generation were obtained by pollinating the silks of T₀transgenic plants with pollen collected from plants of non-transgenicelite inbred line B104, and planting the resultant seeds. Reciprocalcrosses were performed when possible.

Example 5: Biochemical and Molecular Analyses of Transgenic MaizeTissues

ELISA Quantification of AAD1 and PAT Proteins:

Enzyme Linked Immunosorbant Assays (ELISAs) were used to measure theproduction of AAD1 and PAT proteins in maize cells andstably-transformed tissues. AAD1 and PAT proteins were quantified usingkits from ACADIA BIOSCIENCES (Portland, Me.) and ENVIROLOGIX (Portland,Me.), respectively. The ELISAs were performed using multiple dilutionsof plant extracts and using the reagents and instructions essentially asprovided by the suppliers.

Plant Protein Extraction:

Proteins were extracted from 4 leaf discs (totaling 1.3 cm²) or 40immature embryos (for transient expression studies) in 0.6 mL of PBST(PBS buffer containing 0.05% TWEEN® 20) containing either 0.5% BSA (forAAD1 extraction) or 1% PVP-40 (PolyVinylPyrrolidone; for PAT). A 2 mmsteel bead was added, the tubes were capped and secured in aGENO/GRINDER (CERTIPREP; Metuchen, N.J.), and shaken for 5 minutes at1500 rpm. Tubes were centrifuged at 4000 rpm for 7 minutes at 4° C., andsupernatants containing the soluble proteins were stored at −80° C.until used.

Protein extraction from tassels was performed by grinding 0.5 mL tasseltissue in a GENO/GRINDER at 1500 rpm for 2 minutes using garnet powder,one ceramic sphere (0.25 inch diameter; MP BIOCHEMICALS, Santa Anna,Calif.) and 1 mL of PBS containing 5 mM EDTA, 5 mM DTT, 10 μL ProteaseInhibitor Cocktail VI for plant cell research (Research ProductsInternational Corp., Mt. Prospect, Ill.) and 0.4% TWEEN® 20.

Total protein concentrations were determined using a PIERCE 660 nmProtein Assay kit (THERMO SCIENTIFIC; Rockford, Ill.) following thesupplier's instructions.

Hydrolysis Probe qPCR for Copy Number Analysis:

Various types of molecular analyses were employed to screen for lowcopy, simple events. Leaf tissue was collected from rooted putativetransgenic plants before transplanting to soil. DNA was extracted with aQIAGEN MagAttract™ kit using THERMO FISHER KingFisher™ magnetic particleprocessors and the supplier's recommended protocols. Integratedtransgene copy number analysis was performed using specific HydrolysisProbe assays for the AAD1 and PAT genes. In addition, contamination byinadvertent integration of the binary vector plasmid backbone wasdetected by a Hydrolysis Probe assay specific for the Spectinomycinresistance gene (Spec) borne on the binary vector backbone. HydrolysisProbe assays for endogenous maize genes Invertase; (GenBank™ AccessionNo. U16123; SEQ ID NO:14) and Elongation Factor 1α (EF1α) (GENBANKAccession No. AF136823.1; SEQ ID NO:15) were developed as internalreference standards. Table 5 lists the oligonucleotide sequences of theHydrolysis Probe assay components (synthesized by INTEGRATED DNATECHNOLOGIES, Coralville, Iowa). Biplex Hydrolysis Probe PCR reactionswere set up according to Table 6 with about 10 ng DNA, and assayconditions are presented in Table 7.

TABLE 5List of forward and reverse nucleotide primers and fluorescent probes usedfor transgene copy number and relative expression detection. GeneDetected Oligonucleotide Sequence AAD1 AAD1FTGTTCGGTTCCCTCTACCAA (SEQ ID NO: 16) AAD1RCAACATCCATCACCTTGACTGA (SEQ ID NO: 17) AAD1PCACAGAACCGTCGCTTCAGCAACA (SEQ ID NO: 18) (FAM* Probe) PAT_FACAAGAGT GGATTGATGATCTAGAGAGGT PAT (SEQ ID NO: 19) PAT_RCTTTGATGCCTATGTGACACGTAAACAGT (SEQ ID NO: 20) PAT_FamPGGTGTTGTGGCTGGTATTGCTTACGCTGG (FAM Probe) (SEQ ID NO: 21) Spec SPC1ACTTAGCTGGATAACGCCAC (SEQ ID NO: 22) SPC1SGACCGTAAGGCTTGATGAA (SEQ ID NO: 23) TQSPCCGAGATTCTCCGCGCTGTAGA (SEQ ID NO: 24) (FAM Probe) Maize InvertaseFTGGCGGACGACGACTTGT (SEQ ID NO: 25) Invertase InvertaseRAAAGTTTGGAGGCTGCCGT (SEQ ID NO: 26) InvertasePCGAGCAGACCGCCGTGTACTT (SEQ ID NO: 27) (HEX* Probe) Maize EF1a_FATAACGTGCCTTGGAGTATTTGG (SEQ ID NO: 28) Elongation EF1a_RTGGAGTGAAGCAGATGATTTGC (SEQ ID NO: 29) Factor 1α EF1a-MGB*TTGCATCCATCTTGTTGC (SEQ ID NO: 30) (EF1α) (VIC® Probe) *FAM = 6-CarboxyFluorescein Amidite; HEX = Hexachlorofluorescein

TABLE 6 Hydrolysis Probe PCR Mixture for Transgene DNA Copy NumberAnalysis. Reaction Component μL Final Concentration Water 0.5 PVP (10%)0.1 0.1% ROCHE 2X Master Mix 5 1X Transgene Forward Primer (10 μM) 0.40.4 μM Transgene Reverse Primer (10 μM) 0.4 0.4 μM Transgene Probe (5μM) 0.4 0.2 μM Invertase Forward Primer (10 μM) 0.4 0.4 μM InvertaseReverse Primer (10 μM) 0.4 0.4 μM Invertase Probe (5 μM) 0.4 0.2 μM

TABLE 7 Thermocycler Conditions for Hydrolysis Probe PCR Amplification.PCR Steps Temp (° C.) Time No. cycles Denature/Activation 95 10 min 1Denature 95 10 sec 40 Anneal/Extend 58 35 sec Acquire 72  1 sec Cool 4010 sec 1

For amplification, LIGHTCYCLER® 480 Probes Master mix (ROCHE APPLIEDSCIENCE, Indianapolis, Ind.) was prepared at 1× final concentration in a10 μL volume multiplex reaction containing 0.1% PVP, 0.4 μM of eachprimer, and 0.2 μM of each probe. The FAM (6-Carboxy FluoresceinAmidite) fluorescent moiety was excited at 465 nm, and fluorescence wasmeasured at 510 nm. The corresponding values for the HEX(hexachlorofluorescein) fluorescent moiety were 533 nm and 580 nm, andfor VIC® the values were 538 nm and 554 nm. The level of fluorescencegenerated for each reaction was analyzed using the ROCHE LIGHTCYCLER®480 Real-Time PCR system according to the manufacturer'srecommendations. Transgene copy number was determined by comparison ofLIGHTCYCLER 480 outputs of Target/Reference gene values for unknownsamples to Target/Reference gene values of known copy number standards(1-Copy representing hemizygous plants, 2-Copy representing homozygousplants).

Cp scores; i.e., the point at which the florescence signal crosses thebackground threshold using the fit points algorithm (LIGHTCYCLER®software release 1.5), and the Relative Quant module (based on the ΔΔCtmethod), were used to perform the analysis of real time PCR data.

In the LIGHTCYCLER® Fit Points Algorithm software, a graph of the datawas made by plotting the logarithm of the input DNA templateconcentration against the measured Cp values. The slope of the curve isa desired comparison parameter; therefore the initial log input numbercan be an arbitrary starting point on the curve, with the caveat thatthe arbitrary concentration values used for input DNA template arerepresentative of the actual serial dilution used. For example, for a10-fold serial dilution series, the actual inputs concentrations may be1000, 100, 10, etc., for which points the LC480 Fit Points Algorithmsoftware plots 3, 2, 1, etc. as the logarithms of the inputs. Using alinear regression, the resulting best fit of this line (input log vs Cp)was then used to estimate a slope (m) from an equation of the formy=mx+b. There is an inverse relationship between the starting templateamount and Cp value, and therefore the slope (m) is always negative.

A perfect (i.e., 100% efficient) PCR reaction doubles the total templateevery cycle. PCR efficiency (Eff) is calculated as:

Eff=10e ^((−1/m))

Thus, the slope (m) of the graph of log input vs Cp will be −3.3219 fora perfectly efficient reaction (whose efficiency is defined as 2.00).

In other words, a 100% efficient PCR reaction is defined by:

2.0=10e ^((−1/−3.3219))

The LC480 Fit Points Algorithm software reports the efficiency value bythe first formula. So a 99% efficient reaction has an Eff value of 1.99,rather than 0.99.

To express this as a percent efficiency, subtract 1 from this value andmultiply by 100. Or,

% Eff=[(10e ^((−1/m)−1))]×100

AAD1 Relative Transcript Analysis:

Quantitative Hydrolysis Probe PCR was also used to detect the relativelevels of AAD1 transcript. Leaf and tassel tissues were collected at theVT stage (i.e., immediately prior to pollen shed). Approximately 500 ngtotal RNA (extracted with a KingFisher™ total RNA Kit; THERMO FISHERSCIENTIFIC) was used for cDNA synthesis using a high capacity cDNAsynthesis kit (INVITROGEN) and random primer T20VN(TTTTTTTTTTTTTTTTTTTTVN; SEQ ID NO:31, where V is A, C, or G, and N isA, C, G, or T/U). Typically, a 20 μL reaction contained 2.5 U/μL MultiScribe™ reverse transcriptase, 200 nM T20VN oligonucleotide, and 4 mMdNTPs. The reaction was initiated by incubation for 10 minutes at 25°C., then synthesis was performed for 120 minutes at 37° C. andinactivated by 5 minutes at 85° C.

Newly-synthesized cDNA was used for PCR amplification. Hydrolysis ProbeqPCR set up, running conditions, and signal capture were the same asgiven above for DNA copy number analyses. AAD1 expression data werecalculated using 2-ΔΔCt relative to the level of EF1α.

MicroRNA Detection:

Traditional methods such as Northern blot analysis are not suitable forhigh throughput screening of small RNAs, in part due to the requirementfor a large amount of RNA. This study utilized an innovative approachfor detection of mature microRNAs that employed a stem-looped, key-likeprimer for cDNA synthesis, followed by Hydrolysis Probe PCR analysis(Chen et al. (2005) Nucleic Acids Res. 33: e179; Yang et al. (2009)Plant Biotechnol. J. 7: 621-30; Varkonyi-Gasic et al. (2007) PlantMethods 3:12). The key-like RT-PCR primers comprise a universal sequenceof 35 nucleotides on the 5′ end to create a partially double strandedstem-loop structure, and 8 nucleotides on the 3′ end, chosen to becomplementary to the specific mature microRNA, and which are used toinitiate the reverse transcription reaction. This stem-loop structureintroduces a universal reverse PCR primer binding site and a compatibleprobe from a Universal Probe Library (UPL21, ROCHE DIAGNOSTICS) fordownstream PCR amplification, thus alleviating the unique challenge ofdesigning conventional PCR primers to amplify short microRNA sequences.

Total RNA isolated by means of a fully-automated KingFisher™ extractionmethod is sufficient for miRNA detection using Hydrolysis Probe PCRamplification, as well as microRNA analysis. All assays includednegative controls of no-template (mix only). For the standard curves, ablank (water in source well) was also included in the source plate tocheck for sample cross-contamination.

For cDNA synthesis, the key-like primer for miR156 and miR529 (Table 8,miR156_RT and miR529_RT) as well as the oligonucleotides for EF1α(EF1a_F and EF1a_R; Table 5) were included in the reaction (Table 9)using temperature settings of 10 minutes at 25° C. for pre-incubation,120 minutes for synthesis at 37° C., and 5 minutes at 85° C. forinactivation. The RT product was then amplified using amicroRNA-specific forward primer and the universal reverse primer (Table8). FAM-labeled UPL21 was used for fluorescent signal generation andamplification of EF1α was used as an endogenous reference mRNA. ALIGHTCYCLER 480 Real-Time PCR system was used for cycling and signaldetection. Transcription level was calculated using 2-ΔΔCt relative toEF1α. Data were analyzed using LIGHTCYCLER™ Software v1.5 by relativequantification using a second derivative max algorithm for calculationof Cq values according to the supplier's recommendations. For expressionanalyses, expression values were calculated using the ΔΔCt method (i.e.,2-(Cq TARGET—Cq REF)), which relies on the comparison of differences ofCq values between two targets, with the base value of 2 being selectedunder the assumption that, for optimized PCR reactions, the productdoubles every cycle.

TABLE 8 Oligonucleotides used for cDNA Synthesis and miRNADetection (miR156 and miR529). Name Sequence miR156_RTGTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACC AGAGCCAACGTGCTC (SEQ ID NO: 32)miR529_RT GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACAGGCTG(SEQ ID NO: 33) miR156_FGGTGACAGAAGAGAGTGAGCAC (SEQ ID NO: 34) Forward Primer miR529_FGGCGGAGAAGAGAGAGAGTACAG Forward (SEQ ID NO: 35) Primer UniversalGTGCAGGGTCCGAGGT (SEQ ID NO: 36) Reverse Primer UPL21 (FAM*) TGGCTCTG*FAM = 6-Carboxy Fluorescein Amidite

TABLE 9 cDNA Synthesis Mixture for miR156 and miR529. Component μL StockFinal 10X RT Buffer 2 10X 1X miR156_RT Primer 0.5 10 μM 0.25 μMmiR529_RT Primer 0.5 10 μM 0.25 μM EF1a_F Primer 0.5 10 μM 0.25 μMEF1a_R Primer 0.5 10 μM 0.25 μM dNTP mix 0.8 100 mM 4 mM Water 4.2Multiscribe ™ RT 1 50 U/μL 2.5 U/μL RNA 10 Total 20

TABLE 10 Hydrolysis Probe PCR Mixture for miRNA Transcript Detection.Component μL Stock Final 2X Roche MasterMix ™ 5 2X 1X miRNA-SpecificForward Primer 0.4 10 μM 0.4 Universal Reverse Primer 0.4 10 μM 0.4UPL21 0.2 10 μM 0.2 EF1a_F Primer 0.4 10 μM 0.4 EF1a_R Primer 0.4 10 μM0.4 EF1α_MGB VIC ® 0.2 10 μM 0.2 10% PVP 0.1 cDNA 2.0 Water 0.9

Example 6: Tsh4 Target Site-Mediated Reduction of AAD1 mRNA and ProteinProduction

Twenty four hours after particle bombardment of immature embryos withplasmid DNAs containing AAD1 constructs or control plasmids as describedin EXAMPLE 2, the cells were harvested and the amounts of AAD1 proteinproduced were determined by ELISA. Table 11 presents AAD1 proteinproduction data that show elevated amounts of AAD1 protein were producedin cells bombarded with plasmids pDAB113016, pDAB112376, and pDAB112377,compared to cells receiving plasmids pDAB112375, pDAB100286, or notbombarded (Negative Control). Production of AAD1 is repressed in cellstransformed with pDAB112375 (native tsh4 target site), presumably due toa high abundance of miR156 in corn immature embryos. Cells transformedwith plasmids having an AAD1 gene with no miRNA target site(pDAB113016), or with mutated tsh4 target sites (pDAB112377 andpDAB112376), produced higher levels of AAD1 protein. No effect from theartificial miRNA construct harbored by pDAB112378 or from the Cry34Abcontrol construct harbored by pDAB112330 was observed in any of thetreatments. See also FIG. 2.

TABLE 11 AAD1 Protein Levels in Maize Immature Embryos 24 Hours afterParticle Bombardment. Mean AAD1 Co-bombardment Amount AAD1 Test Plasmidplasmid (ng/mL) pDAB113016 (no miRNA target site) pDAB112330 302(Cry34Ab) pDAB113016 (no miRNA target site) pDAB112378 436 (artificialmiRNA) pDAB112376 (SEQ ID NO: 4; pDAB112378 317 modified tsh4 targetsite) (artificial miRNA) pDAB112377 (SEQ ID NO: 6; pDAB112378 249modified tsh4 target site) (artificial miRNA) pDAB112375 (SEQ ID NO: 2;native pDAB112378 51 tsh4 target site) (artificial miRNA) pDAB112375(SEQ ID NO: 2; native pDAB112330 58 tsh4 target site) (Cry34Ab)pDAB100286 (No AAD1; YFP) None 0.00 None (Negative Control) None 0.00

Agrobacterium-mediated procedures were used to producestably-transformed maize immature embryos using plasmids pDAB113018,pDAB113019, pDAB113020, and pDAB113021. Table 4. Plasmid pDAB113018harbors two selectable marker genes (PAT and AAD1) within the T-DNAborders (EXAMPLE 2). The mRNA produced from the AAD1 transgene inpDAB113018 contains a native tsh4 miR156/miR529 target site sequence.pDAB113018-transformed embryos were randomly split between plates ofSelection Medium I containing 100 nM R-Haloxyfop acid (for selection oftransformants harboring the AAD1 gene) or 5.0 mg/L Bialaphos (forselection of transformants harboring the PAT gene). All the embryosplaced on Haloxyfop-containing medium browned and died, while theembryos on Bialaphos-containing medium grew and regenerated as usual.Transgenic embryos are routinely selected, grow and regenerate onHaloxyfop-containing medium when transformed with an AAD1 gene lackingthe tsh4 target site sequence. The immature embryos transformed withplasmids having an AAD1 gene with no miRNA target site (pDAB113016), orwith mutated tsh4 target sites for miR156 (pDAB112377 and pDAB112376),produced higher levels of AAD1 protein grew on Haloxyfop-containingmedium and regenerated transgenic plants. Thus, levels of AAD1 proteinproduced in pDAB113018-transformed cells are insufficient to provideHaloxyfop tolerance. Death of the pDAB113018-transformed embryos onHaloxyfop-containing medium is presumably due to destruction of the AAD1mRNA in the developing embryos by miR156-mediated mRNA cleavage.

Stable transgenic T₀ plants (1 to 2 copies of the transgenes) weretransferred to the greenhouse for mature plant production. For eachconstruct, 8 to 12 T₀ plants were tested for leaf expression of AAD1mRNA and protein, and 4 to 6 T₀ plants were tested for tassel expressionof AAD1 mRNA and protein. AAD1 mRNA levels were calculated relative tothe levels of maize Elongation Factor mRNA as described in EXAMPLE 5.Table 12 shows the results of the analyses. See also FIGS. 3A-5B.Examination of the data shows that there is up to a 6-fold reduction ofmRNA production and an 11-fold repression of AAD1 protein production intassel tissues in plants transformed with plasmids expressing AAD1 mRNAcontaining an miR529 target site (plasmids pDAB113018, pDAB113019, andpDAB113020), when compared to tissues producing AAD1 mRNA with no targetsite. In leaf tissues, however, there is very little difference ineither AAD1 mRNA or protein accumulation between plants transformed withthe different plasmids. This result is in line with the data of Table 1,which shows that the level of miR529 is 16- to 100-fold higher inimmature tassel tissues than in leaves. Thus, the results summarized inTable 11, and particularly in Table 12, demonstrate that engineering ofmRNA to include miRNA target cleavage sites may be used as a mechanismto control tissue-specific accumulation of a specific mRNA and protein.

TABLE 12 Levels of AAD1 mRNA and Protein in Leaves and Tassels ofTransgenic B104 Plants. Means were separated using the Tukey-KramerTest. Leaves Tassels Transforming mRNA mRNA Plasmid Level* Protein**Level* Protein** pDAB113018 0.371 (A)*** 110.22 (B) 0.420 (B)  145.86(B) pDAB113019 0.261 (A) 140.10 (AB) 0.752 (B)  341.54 (B) pDAB1130200.294 (A) 117.37 (AB) 0.760 (B)  190.30 (B) pDAB113021 0.340 (A) 201.45(A) 2.423 (A) 1730.50 (A) (no target site) *Relative to maize EF1α mRNA.**ng AAD1 protein/mg Total Soluble Protein ***Levels not connected bythe same letter are significantly different

A transgenic maize plant is produced wherein the accumulation of an AAD1mRNA comprising an miRNA target site is limited by cleavage by miRNA(e.g., miR156, miR529, and miR319) in tissues or cells comprising theimmature tassel, including the palea, lemma, stamen, filament, anther,microspores, male gametophyte, sperm cell, tube cell, or other cellsthat affect or control pollen development. Such limited accumulation ofAAD1 mRNA is insufficient to produce a level of AAD1 proteinaccumulation sufficient to confer tolerance of immature tassel tissuesto herbicides that are substrates for AAD1, such as Haloxyfop andQuizalofop. Thus, treatment of such transgenic plants at an immaturetassel stage of development with herbicides that are substrates for AAD1prevents tassel and pollen development, and results in a male sterileplant. Micro RNA expression profiling disclosed in Table 1 shows thatmiR529 is not specifically expressed in the tassel tissue, but rather isexpressed at similar levels in both tassel and ear tissues in laterstages of development. Therefore, treatment of transgenic plants(accumulating AAD1 mRNA comprising said miRNA target site) withherbicides that are substrates for AAD1 during an early stage of tasseldevelopment prevents tassel and pollen development, thus resulting inmale sterility, and without affecting ear tissue development.

Example 7: AAD1 mRNA and Protein Production in T₁ Maize Plants

Five single copy transgenic events per construct were planted in thegreenhouse for T1 expression analysis. For each event; 7 to 8 plants(35-40 plants per construct) were tested for leaf expression of AAD-1mRNA, and 2 plants (10 plants per construct) were tested for expressionof AAD-1 mRNA and protein in different tissues in multiple growthstages. AAD-1 mRNA levels were calculated relative to the levels ofmaize Elongation Factor mRNA (EF1α transcripts) as described in EXAMPLE5. The data in Table 13 show that there was no effect of miRNA targetsites on AAD1 expression in leaves of V6 plants transformed withconstructs pDAB113019 and pDAB113020 compared to the “no miRNA” controlplants transformed with pDAB113121. However, a significant decrease inAAD1 mRNA expression was observed in plants transgenic to constructspDAB113018 that contain native miRNA target site without any mutation.The AAD1 reduction in these plants is potentially due to miR156 that ispresent in high abundance in the leaf tissue. These results confirm thatmutations for the miR156 site made in pDAB113019 and pDAB113020 removedthe miR156 binding in these constructs and therefore down regulation ofAAD1 was avoided in the leaf tissue.

TABLE 13 Ratio of AAD1 to EF1α mRNA levels in Leaves of T₁ TransgenicB104 Plants at the V6 Growth Stage. Means were separated using theTukey-Kramer Test. Transforming Number of Mean mRNA Plasmid plantssampled Ratio pDAB113021 38 2.31 (A)* pDAB113020 40 2.30 (A) pDAB11301940 2.09 (A) pDAB113018 37 1.30 (B) *Levels not connected by same letterare significantly different.

The protein and RNA expression analysis of immature tassel in V8 stageshowed significant down regulation of both AAD1 mRNA and protein intransgenic plants expressing AAD-1 mRNA containing an miR529 target site(plasmids pDAB113018, pDAB113019, and pDAB113020), when compared totissues producing AAD-1 mRNA with no target site (pDAB113021). (Table14) There was a similar level of AAD1 reduction in plants transformedwith pDAB113018 and pDAB113020, which confirms that AAD1 down regulationin immature tassel was due to miR529 and mutation made in the miRNAbinding site of construct pDAB113020 did not affect the binding ofmiR529. There was significant but limited down regulation of AAD1 in theplants transformed with pDAB113019, indicating that mutation in themiRNA binding site of construct pDAB113019 had some effect on miR529binding. These results confirm that desired and precise down regulationof the transgene could be obtained by selected manipulations of bindingsites of native miRNA. In leaf tissues of V8 plants, however, there wasvery little difference in either AAD-1 mRNA or protein accumulationbetween plants transformed with pDAB113019 and pDAB113020 containing amodified miR529 target site (Table 14).

TABLE 14 Ratio of AAD1 to EF1α mRNA levels, and level of AAD1 Protein,in Tassels and Leaves of T₁ Transgenic B104 Plants at the V8 GrowthStage. Means were separated using the Tukey-Kramer Test. Tassel LeafTransforming Mean mRNA Mean mRNA Plasmid Ratio Protein** Ratio Protein**pDAB113021 0.84 (A)* 339 (A) 0.86 (A) 107.5 (A) (no target site)pDAB113019 0.54 (AB) 188 (B) 0.79 (AB) 140.8 (A) pDAB113020 0.33 (B) 49.8 (C) 0.96 (A)  91.1 (A) pDAB113018 0.31 (B)  47.1 (C) 0.45 (B) 74.5 (A) *Levels not connected by same letter are significantlydifferent. **ng/mg total protein

Similarly, there was slight or no down regulation of AAD1 in theimmature ear in V12 stage plants, or in the leaves of R3 stage plants,transformed with the various constructs (Table 15). However, compared tothe control “no miRNA target site” construct pDAB113021, there was asubstantial reduction of AAD1 protein in the V6 root tissue of plantstransformed with pDAB113018, pDAB113019, and pDAB113020 (Table 15).These results confirm that hat miR529 is not specifically expressed inthe tassel tissue, but rather is expressed at some levels in V6 rootstage resulting in substantial reduction of AAD1 protein.

TABLE 15 Levels of AAD1 Protein (ng/mg total protein) in SelectedTissues of T₁ Transgenic B104 Plants at Three Growth Stages. Means wereseparated using the Tukey-Kramer Test. Transforming Immature ear,Plasmid V12 stage Leaf, R3 stage Root, V6 stage pDAB113021 312.7 (AB)112.8 (AB)* 163.6 (A) pDAB113020 256.3 (B) 119.4 (AB) 102.9 (BC)pDAB113019 296.3 (AB) 137.6 (A) 137.4 (AB) pDAB113018 408.9 (A)  74.7(B)  51.4 (C)

Example 8: Herbicide-Induced Control of Pollen Production and MaleSterility

Five transgenic maize T₀ events of line B104 were produced byAgrobacterium-mediated transformation with plasmid pDAB109812,essentially as provided in EXAMPLE 4, and grown in the greenhouse. Nullnegative control plants (no AAD1 gene) and isoline positive controlplants (in which AAD1 mRNA expression was driven by a copy of the maizeubiquitin 1 promoter with associated intron 1, and terminated by a copyof a maize per5 3′UTR, both as described above) were planted at the sametime.

Thirty T₁ plants (derived by fertilization of the 5 T₀ events withB104-derived pollen) were grown in the greenhouse and subjected to aselection spray of 70 g acid-equivalents per hectare (ae/ha) of Assure®II at about the V2 stage to remove null segregant plants and providehemizygous plant survivors having a functional AAD1 gene. Assure® IIcontains active ingredient Quizalofop P-EthylEthyl(R)-2-4-[4-6-chloroquinoxalin-2-yl oxy)-phenoxy]propionate, and isproduced by Dupont™ Crop Protection, Wilmington, Del. The commercialproduct contains 0.88 lbs. active ingredient (ai) per gallon.)

Twelve hemizygous plants for each event were chosen, and at the time oftassel appearance the whorls were treated according to Table 16.Controls were treated in the same way. Plants were grown to maturity inindividual 5-gallon pots. When the tassel was partially emerged, leavessurrounding the tassel were carefully removed by trimming. Tassels weresprayed with either a 1.143% (v/v) or 2.286% (v/v) of Assure® IIsolution containing 1.0% (v/v) crop oil concentrate (COC). COC is anemulsifiable refined paraffinic oil containing about 85% paraffinic oiland about 15% emulsifiers. Applications were accomplished by applying 8bursts of spray (0.1 mL each) from a hand-held DeVilbiss bulb atomizer.This method distributed 0.8 mL treatment solution to the tassel, andprovides the equivalent of 560 gm ai/ha and 1120 gm ai/ha, respectively,for the 1.143% and 2.286% solutions. Neighboring plants and tissues wereshielded from spray drift.

TABLE 16 Distributions of treatments amongst 12 hemizygous T1 plantsfrom transgenic B104 events previously selected for a functional AAD1gene (n = 12). Treatment* No. Plants Treated 0 (untreated) 2 0 (1% COCCheck) 2  560 4 1120 4 *g ae/ha Assure ® II

Tassel damage was characterized by visual assessment of tissue death asa percentage of total tassel tissue.

Leaf paint assessments were performed using the same solutions as wereused on the tassel sprays. Leaf paints were applied on both sides of themidvein (without contacting the midvein) of a 4-inch segment located inthe middle of the 4^(th) oldest leaf, using a small sponge paintapplicator.

Tassels, leaves and stalks were rated for injury after applications(Table 17 and Table 18). There was 100% tassel elimination for plants offour out of the five pDAB109812 (i.e., SCBV:AAD1) events, effectivelyinducing male sterility. Additionally, leaves and stalks of these plantswere not damaged. Thus, the plants retained effective herbicidetolerance in the vegetative and female reproductive tissues. Thepositive control plants (maize ubiquitin1 promoter driving AAD1expression) did not show any tassel or vegetative injury, demonstratingtissue-specific AAD1 expression with the SCBV promoter. The null controlplants (no AAD1 gene) showed 100% tassel elimination and also vegetativeinjury, as expected.

TABLE 17 Summary of event data. % Tassel % Tassel Leaf Paint InjuryInjury Stalk Injury Event Plant Treatment* 12 DAA** 20 DAA 20 DAA109812[1]-019.001AJ 1 0 (untreated) 0  0 NA** 2 0 (untreated) 0  0 NA 3560 30 30 No 4 560 20 30 No 5 0 (COC check) 0  0 No 6 560 20 30 No 7 56020 30 No 8 1120 30 30 No 9 1120 10 20 No 10 1120 30 70 No 11 1120 10 20No 12 0 (COC check) 0  0 No 109812[1]-009.001AJ 1 0 (untreated) 0  0 2 0(untreated) 0  0 3 0 (COC check) 0  0 No 4 0 (COC check) 0  0 No 5 56030  100*** No 6 560 40  100*** No 7 560 30 30 No 8 560 30 30 No 9 112040 40 No 10 1120 30  100*** No 11 1120 30 30 No 12 1120 30 30 No109812[1]-005.001AJ 1 0 (untreated) 0  0 2 560 40 50 No (delayed) 3 0(COC check) 0  0 No 4 560 40  100*** No 5 560 30 30 No 6 560 30 40 No 70 (COC check) 30  0 No 8 1120 30  100*** No 9 1120 30 30 No 10 1120 30 100*** No 11 1120 30 30 No 12 NT** NT NT NT 109812[1]-010.001AJ 1 0(untreated) 0  0 2 0 (untreated) 0  0 3 560 30 30 No 4 560 40 40 No 5 0(COC check) 0  0 No 6 560 30 30 No 7 1120 90  100*** Yes (ear only) 8 0(COC check) 0  0 No 9 560 50 95 No 10 1120 30 30 No 11 1120 30 30 No 121120 50 40 No 109812[1]-018.001AJ 1 0 (untreated) 0  0 2 0 (COC check) 0 0 No 3 0 (COC check) 0  0 No 4 560 30 85 No 5 560 30 30 No 6 560 0  0No 7 1120 30 30 No 8 560 50 100† No 9 1120 30 30 No 10 1120 20 50 No 111120 20 30 No 12 NT NT NT *g ai/ha Assure ® II **DAA = Days AfterApplication; NA = Not Applicable; NT = Not Tested ***Tassel was necroticand did not expand †Tassel and top leaves died

TABLE 18 Summary of control plant data. % Tassel Leaf Paint % Tassel %Tassel Leaf Paint Injury Symptoms Injury Injury Stalk Injury Source IDTreatment* 7 DAA 7 DAA 12 DAA 22 DAA 22 DAA Null (No AAD1 gene) 1  56030 Yes 70 100 Yes 2  560 20 Yes 50 100 Yes 3  560 40 No 60 100 Yes 4 560 50 Yes 70 100 Yes 5 1120 40 No 70 100 Yes 6 Untreated 0 0 0 No 71120 50 Yes 70 100 Yes 8 1120 20 No 30 90 Yes 9 Untreated 0 0 0 No 10Untreated 0 0 0 No 11 1120 30 Yes 50 90 Yes 12 Untreated 0 0 0 NoPositive control (High AAD1 expression) 1  560 0 Yes 0 0 No 2  560 0 Yes0 0 No 3  560 0 Yes 0 0 No 4  560 0 No 0 0 No 5 Untreated 0 0 0 No 6Untreated 0 0 0 No 7 1120 0 Yes 0 0 No 8 1120 0 Yes 0 0 No 9 Untreated 00 0 No 10 1120 0 No 0 0 No 11 Untreated 0 0 0 No 12 1120 0 Yes 0 0 No *gai/ha Assure ® II **DAA = Days After Application; NA = Not Applicable;NT = Not Tested ***Tassel was necrotic and did not expand †Tassel andtop leaves died

In summary, the data show that a timed and directed application ofAssure® II to SCBV:AAD1 plants can effectively kill male reproductivetissue, whilst still providing vegetative tolerance.

Example 9: Herbicide-Induced Male Sterile Hybridization System

Transgenic maize plants are produced that have been transformed with anAAD1 gene under the control of a plant promoter and other plantexpression regulatory elements. Inherent to the AAD1 mRNA is a targetsite sequence recognized, for example, by an miRNA (e.g., miR156,miR529, and miR319). It is advantageous that native expression of themiRNA whose target site is introduced into the AAD1 mRNA is detected intissues or cells comprising the immature tassel, including the palea,lemma, stamen, filament, anther, microspores, male gametophyte, spermcell, tube cell, or other cells that affect or control pollendevelopment, although expression may also be detected in ear and roottissue (Chuck et al. (2010) Development 137:1243-50; Zhang et al. (2009)PloS Genetics November 5).

It is additionally advantageous that the promoter used to controlexpression of the AAD1 mRNA be one that is inactive or only weaklyfunctional in tissues or cells comprising the immature tassel, includingthe palea, lemma, stamen, filament, anther, microspores, malegametophyte, sperm cell, tube cell, or other cells that affect orcontrol pollen development and production.

It is further advantageous that expression of AAD1 mRNA in other plantparts not directly involved in pollen production (such as leaves, roots,stems, developing ears, silks, etc.) is sufficient to produce an AAD1protein accumulation to levels sufficient to confer tolerance of thosetissues to growth inhibitory levels of herbicidal compounds that aresubstrates for AAD1, such that those tissues are not damaged orotherwise detrimentally affected by application (for example, byspraying) of the herbicide compounds. Application of inhibitory levelsof herbicidal compounds that are substrates for AAD1 to plants at anearly stage of tassel development inhibits or prevents tassel and pollendevelopment and thus produces a male sterile plant. Further, thedevelopment, growth, morphology, maturity and yield of such plants arenot affected by treatment with the herbicidal compounds.

Example 10: Stereospecific Herbicide-Induced Male Sterile HybridizationSystems

Transgenic maize plants having an AAD1 transgene are produced, in whichexpression of the AAD1 transgene is under the control of constitutiveplant expression regulatory elements, such as the maize ubiquitin1promoter with associated intron 1. Such AAD1 plants are secondarilytransformed with a plant expression cassette comprising an AAD12 codingregion (U.S. Pat. No. 8,283,522) under the control of a promoter andother plant expression regulatory elements that are chosen such thatAAD12 transgene expression and accumulation of the AAD12 protein occurexclusively in male reproductive tissues (for example, immature tassel,palea, lemma, stamen, filament, anther, microspores, male gametophyte,sperm cell, tube cell, or other cells that affect or control pollendevelopment and production).

The AAD1 and AAD12 proteins utilize enantiomeric substrates (the R-formor the S-form, respectively) of compounds comprising a benzene ring,oxygen, and propionic acid. For example, the AAD12 protein can use assubstrates 2,4-D (2,4-dichlorophenoxyacetic acid) and pyridyloxyacetateherbicidal compounds, whereas the AAD1 protein inactivates “fops,”substrates such as Haloxyfop and Quizalofop. During hybrid seedproduction female rows (containing plants to be used as pollenacceptors) are sprayed at an early tassel stage (before pollen shed)with an herbicidal compound analog (pro-herbicide S-form) that is aspecific substrate for AAD12, whilst the male rows (containing plants tobe used as pollen donors) are not sprayed.

The activity of AAD12 protein in the male tissues of the sprayed plantsconverts the S-form pro-herbicidal compound into an active herbicidalcompound, which results in destruction of the male tissues and thusinduces male sterility. The other tissues of the sprayed plants (as aresult of AAD1 protein production and the lack of AAD12 production inthose tissues) are not affected by the S-form pro-herbicidal compoundand are further resistant to fops herbicidal compounds (for example,Haloxyfop and Quizalofop). Thus, weeds in the fields are controlled bythe action of the fops herbicides. Subsequently, plants in unsprayedmale rows are able to produce pollen and to pollinate plants in femalerows to create hybrid seed. The method avoids yield loss due to plantdamage that occurs during a machine or manual de-tasseling process,which becomes unnecessary when a herbicide-induced male sterility systemis employed.

In some examples, an additional herbicide tolerance gene (such as a DGTgene (U.S. Patent Application 61/593,555, filed Feb. 1, 2012)) isexpressed throughout the whole plant, while an AAD12 gene is expressedexclusively in the male tissues, with the same end result of inductionof male sterility upon application of the appropriate herbicidalcompounds at the appropriate stages of plant development. Otherherbicide tolerance genes are used in some examples.

In dicotyledonous plants, the roles of the AAD1 and AAD12 proteins isreversed, such that AAD12 would be constitutively expressed throughoutthe plant, while AAD1 expression would be male-tissue specific, and anR-form pro-herbicidal compound would be used to destroy male tissues.

What may be claimed is:
 1. A nucleic acid molecule comprising a geneoperably linked to a plant promoter, wherein the gene comprises theinternal small ribonucleic acid (sRNA) target site of SEQ ID NO:7. 2.The nucleic acid molecule of claim 1, wherein the molecule is a vector.3. The nucleic acid molecule of claim 2, wherein the vector is a plantexpression vector or plant transformation vector.
 4. The nucleic acidmolecule of claim 1, wherein the molecule is a plant genomic nucleicacid molecule.
 5. The nucleic acid molecule of claim 1, wherein the geneis an herbicide tolerance gene or a selectable marker gene.
 6. Thenucleic acid molecule of claim 1, wherein the plant promoter issugarcane bacilliform virus (SCBV) promoter.
 7. A transgenic plant cellcomprising the nucleic acid molecule of claim
 1. 8. The transgenic plantcell of claim 7, wherein expression of the gene is reduced or eliminatedin at least one tissue of the plant.
 9. The transgenic plant cell ofclaim 7, wherein expression of the gene is reduced or eliminated in atissue-specific or tissue-preferred manner.
 10. The transgenic plantcell of claim 9, wherein expression of the gene is reduced or eliminatedin male plant tissues.
 11. A transgenic plant tissue or plant partcomprising the nucleic acid molecule of claim
 1. 12. The transgenicplant tissue or plant part of claim 11, wherein expression of the geneis reduced or eliminated in male plant tissues.
 13. The transgenic planttissue or plant part of claim 11, wherein the plant tissue or plant partis a seed.
 14. A transgenic plant comprising the nucleic acid moleculeof claim
 4. 14. The transgenic plant of claim 14, wherein expression ofthe gene is reduced or eliminated in at least one tissue of the plant.16. The transgenic plant of claim 15, wherein expression of the gene isreduced or eliminated in a tissue-specific or tissue-preferred manner.17. The transgenic plant of claim 16, wherein expression of the gene isreduced or eliminated in male plant tissues.
 18. The transgenic plant ofclaim 17, wherein the gene is an herbicide tolerance gene, and whereinapplication of the herbicide to the plant kills male tissues in theplant.
 19. A method for producing a transgenic plant cell, the methodcomprising transforming a plant cell with the nucleic acid molecule ofclaim
 1. 20. The method according to claim 19, the method furthercomprising: culturing the transformed plant cell to produce a plant cellor tissue culture of transgenic plant cells comprising the gene operablylinked to the plant promoter; and regenerating a plant from the plantcell or tissue culture.